WO2024046393A1 - Procédé de trans-différenciation de cellules non neuronales en neurones et son utilisation - Google Patents

Procédé de trans-différenciation de cellules non neuronales en neurones et son utilisation Download PDF

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WO2024046393A1
WO2024046393A1 PCT/CN2023/115930 CN2023115930W WO2024046393A1 WO 2024046393 A1 WO2024046393 A1 WO 2024046393A1 CN 2023115930 W CN2023115930 W CN 2023115930W WO 2024046393 A1 WO2024046393 A1 WO 2024046393A1
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cells
neurons
gene
aav
neuronal
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PCT/CN2023/115930
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Chinese (zh)
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周海波
胡新德
苏锦霖
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上海鲸奇生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present application relates to the field of translational medicine, and more specifically, to a method for transdifferentiating non-neuronal cells into neurons, and the use of this method for preparing drugs for the treatment or prevention of diseases related to neuronal damage or neuron death.
  • the main pathological changes caused by nervous system damage and various neurodegenerative diseases are neuronal degeneration, necrosis and neuronal damage. Since the self-repair ability of the nervous system (such as the brain, spinal cord, optic nerve, etc.) is very limited, it is difficult to repair neuron cells autonomously. Diseases related to neuronal function loss or neuron death have always been difficult to treat, such as Parkinson's. disease, schizophrenia, depression, Alzheimer's disease, Huntington's disease, sleep disorders, brain trauma, stroke, visual system diseases related to loss or death of RGC or photoreceptor cell function, blindness, deafness, etc., the current treatment methods are only It is to alleviate the progression of the disease, but there has been a lack of effective treatments.
  • Cell transdifferentiation refers to the process by which one type of differentiated cells can structurally and functionally transform into another type of differentiated cells under certain conditions. If some non-neuronal cells in the nervous system can be reprogrammed into neuronal cells, it will be expected to fundamentally treat diseases related to neuronal function loss or neuron death.
  • Another object of the present disclosure is to provide one or more inhibitors of the expression or activity of genes or their RNAs or their encoded proteins selected from the group consisting of for preventing and/or treating neuronal function loss or death associated with Disease uses: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B.
  • Another object of the present disclosure is to provide one or more enhancers of the expression or activity of genes or their RNAs or their encoded proteins selected from the group consisting of for preventing and/or treating neuronal function loss or death associated with Disease uses: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC.
  • the present disclosure provides a method of generating neuronal cells from glial cells, comprising using One or more inhibitors of the expression or activity of genes or their RNA or their encoded proteins transdifferentiate or reprogram the glial cells into neurons selected from: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b , HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, wherein the inhibitor reduces the expression or activity of the gene or its RNA or its encoded protein, and the method includes :
  • the present disclosure provides a method of generating neuronal cells from glial cells, comprising using one or more enhancers of the expression or activity of a gene or its RNA or its encoded protein selected from: Transdifferentiating or reprogramming the glial cells into neurons: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC, wherein the enhancer increases the expression of the gene or its RNA or its encoded protein Or activity, the method includes:
  • the glial cells are mammalian glial cells, including glial cells of human, non-human primate, mouse, and rat species.
  • the mammalian glial cells include astrocytes, oligodendrocytes, microglia, NG2 cells, Müller glia, glioma cells or spiral nerves Glial cells.
  • the glial cells are astrocytes or Müller glia.
  • the astrocytes are derived from the brain, midbrain, cerebellum, brainstem, and spinal cord.
  • the astrocytes are derived from the striatum or substantia nigra.
  • the Müller glia cells are derived from the retina.
  • the spiral ganglion glial cells are derived from the inner ear or vestibule.
  • the neuronal cells are mammalian neurons, including neurons of humans, non-human primates, rats, and mice.
  • neuronal cells are selected from dopamine neurons, 5-HT neurons, NE neurons, ChAT neurons, motor neurons, GABA neurons, glutamatergic neurons, Spinal cord neurons, spinal motor neurons, spinal sensory neurons, photoreceptor cells (rods and cones), bipolar cells, amacrine cells, Retinal ganglion cells (RGC), cochlear nerve cells, pyramidal neurons, interneurons, medium spiny neurons (MSN), Purkinje cells, granule cells, olfactory sensory neurons, periglomerular cells or combinations thereof, more preferably dopamine neurons, retinal ganglion cells and photoreceptor cells.
  • dopamine neurons 5-HT neurons, NE neurons, ChAT neurons, motor neurons, GABA neurons, glutamatergic neurons, Spinal cord neurons, spinal motor neurons, spinal sensory neurons, photoreceptor cells (rods and cones), bipolar cells, amacrine cells, Retinal ganglion cells (RGC), cochlear nerve cells, pyramidal neurons, interneurons, medium spiny neurons (MSN
  • said glial cells are astrocytes; and said neuronal cells are dopamine neurons.
  • said glial cells are Müller glia; and said neuronal cells are RGCs or photoreceptor cells.
  • the present disclosure provides the use of one or more inhibitors of the expression or activity of genes or their RNA or their encoded proteins selected from the group consisting of: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, the medicine is used to prevent and/or treat diseases related to neuronal function loss or death, wherein The inhibitor reduces the expression or activity of the gene or its RNA or its encoded protein.
  • the present disclosure provides the use of one or more enhancers of the expression or activity of genes or their RNA or their encoded proteins selected from the group consisting of: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC, the medicine is used to prevent and/or treat diseases related to neuronal function loss or death, wherein the enhancer increases the expression of the gene or its RNA or its encoded protein or active.
  • the medicament is formulated for in vivo administration to the nervous system, visual system, and auditory system, including the striatum, substantia nigra, midbrain ventral tegmental area, spinal cord, hypothalamus, dorsal Lateral midbrain, cerebral cortex, hippocampus, cerebellum, subretinal, vitreous cavity, inner ear cochlea and vestibule.
  • the medicament is formulated for administration to the striatum, substantia nigra, subretina and vitreous cavity.
  • the disease associated with neuronal function loss or death is a neurological disease, including Parkinson's disease, schizophrenia, depression, Alzheimer's disease, Huntington's disease, epilepsy, sleep disorders disorders, ataxia, PloyQ disease, cerebral ischemia, brain injury, addiction, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, Pick's disease, associated with loss of function or death of RGC or photoreceptor cells visual system diseases, blindness, and deafness.
  • a neurological disease including Parkinson's disease, schizophrenia, depression, Alzheimer's disease, Huntington's disease, epilepsy, sleep disorders disorders, ataxia, PloyQ disease, cerebral ischemia, brain injury, addiction, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, Pick's disease, associated with loss of function or death of RGC or photoreceptor cells visual system diseases, blindness, and deafness.
  • the diseases related to neuron function loss or death are Parkinson's disease and visual system diseases related to RGC or photoreceptor cell function loss or death.
  • the present disclosure provides a method of generating retinal ganglion cells (RGC) or photoreceptor cells from Müller glia, comprising using one or more genes or RNA thereof selected from or Inhibitors of the expression or activity of the encoded proteins enable the Müller glia to transdifferentiate or reprogram into RGCs or photoreceptor cells: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, wherein the inhibitor reduces the gene or its RNA or its encoded protein White expression or activity.
  • RGC retinal ganglion cells
  • the present disclosure provides a method of generating retinal ganglion cells (RGC) or photoreceptor cells from Müller glia, comprising using one or more genes or RNA thereof selected from or The enhancer of the expression or activity of its encoded protein causes the Müller glial cells to transdifferentiate or reprogram into RGCs or photoreceptor cells: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC, wherein the enhancer The agent increases the expression or activity of the gene or its RNA or its encoded protein.
  • RGC retinal ganglion cells
  • the enhancer of the expression or activity of its encoded protein causes the Müller glial cells to transdifferentiate or reprogram into RGCs or photoreceptor cells: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC, wherein the enhancer The agent increases the expression or activity of the gene or its RNA or its encoded protein.
  • the Müller glia cells are derived from the retina.
  • the photoreceptor cells include rods and cones.
  • the present disclosure provides the use of one or more inhibitors of the expression or activity of genes or their RNA or their encoded proteins selected from the group consisting of: RCOR1, RCOR2, RCOR3, Sin3a in the preparation of a medicament.
  • the drug is used to prevent and/or treat the visual system related to RGC or photoreceptor cell function loss or death Diseases wherein the inhibitor reduces the expression or activity of the gene or its RNA or its encoded protein.
  • the present disclosure provides the use of one or more enhancers of the expression or activity of genes or their RNA or their encoded proteins selected from the group consisting of: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC, the drug is used to prevent and/or treat visual system diseases related to RGC or photoreceptor cell function loss or death, wherein the enhancer increases the gene or its RNA or its coding protein expression or activity.
  • the medicament is formulated for administration to the visual system.
  • the drug is formulated for subretinal or intravitreal use, wherein the drug acts by acting on Müller glia.
  • the neurological disease related to RGC function loss or death is selected from: vision impairment caused by RGC cell death, glaucoma, age-related RGC lesions, diabetes-related retinopathy, optic nerve damage, retinal defects Blood or hemorrhage, Leber hereditary optic neuropathy, or combinations thereof.
  • the visual system disease related to photoreceptor cell function loss or death is selected from: photoreceptor cell degeneration or death caused by injury or degenerative disease, macular degeneration, retinitis pigmentosa, diabetes-related blindness, Night blindness, color blindness, hereditary blindness, congenital amaurosis, or combinations thereof.
  • the inhibitor or agonist is selected from: antibodies, small molecule compounds, mRNA, microRNA, siRNA, shRNA, antisense oligonucleotides, binding proteins or protein domains, polypeptides, nucleic acid adapters Ligands, gene editors, PROTACs, expression vectors containing promoters, endogenous expression activators, protein analogs or enhancers, synthetic or modified inhibitors or enhancers mentioned above, or combinations thereof.
  • the present disclosure provides a pharmaceutical composition or kit or kit comprising one or more inhibitors of the expression or activity of a gene or its RNA or its encoded protein selected from : RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B; and/or one or more genes selected from the following or their RNA or their encoding Enhancers of protein expression or activity: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC.
  • a gene or its RNA or its encoded protein selected from : RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B
  • the inhibitor comprises:
  • gRNAs or expression vectors thereof wherein the gRNA is DNA or RNA that guides the gene editing protein to specifically bind to the gene.
  • the editing system includes: CRISPR system (including CRISPR/dCas system), ZFN system, TALEN system, RNA editing system, or a combination thereof.
  • the gene editing protein is an RNA-targeting gene editing protein.
  • the gRNA is an RNA-targeting gRNA.
  • said enhancer comprises: an expression vector containing a promoter, an endogenous expression activator, a protein analog or an enhancer.
  • the pharmaceutical composition or kit further comprises a vehicle for delivering the inhibitor.
  • the carrier is a viral vector, liposome, nanoparticle, exosome, virus-like particle, preferably AAV.
  • the RNA-targeting gene editing protein is selected from the group consisting of: Cas13d, Cas13e, Cas13a, Cas13b, Cas13c, Cas13f and their functional domains.
  • the RNA-targeting gene editing protein is selected from the group consisting of: CasRx, Cas13e, and Cas13f.
  • RNA-targeting gene editing protein is CasRx.
  • said pharmaceutical composition or kit or kit contains only a single type of gRNA targeting said mRNA sequence or 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12 different gRNAs.
  • the gRNA expression vector encodes a gRNA comprising only a single type of gRNA targeting the mRNA sequence or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 different gRNAs.
  • the expression vector contains:
  • nucleotide sequence encoding the gene editing protein operably linked to a promoter causing expression of the gene editing protein
  • the promoter is a broad spectrum promoter or a specific promoter.
  • the broad-spectrum promoter is selected from the group consisting of CMV, CBH, CAG, PGK, SV40, EFlA, EFS, and pGlobin promoters.
  • the specific promoter is a glial cell-specific promoter or a Müller glial (MG) cell-specific promoter.
  • the glial cell-specific promoter is selected from the group consisting of GFAP promoter, ALDH1L1 promoter, EAAT1/GLAST promoter, glutamine synthetase promoter, S100 ⁇ promoter and EAAT2/GLT- 1 promoter, NG2 promoter, CD68 promoter, F4/80 promoter, or the MG cell-specific promoter is selected from the group consisting of GFAP promoter, ALDH1L1 promoter, Glast (also known as Slc1a3) promoter and Rlbp1 promoter .
  • the expression vector is contained in a viral particle.
  • the expression vector is a gene therapy vector.
  • the gene therapy vector is a viral gene therapy vector.
  • the expression vector is a viral vector selected from the group consisting of: adeno-associated virus (AAV) vector, recombinant adeno-associated virus vector (rAAV), self-complementing adeno-associated virus vector (scAAV), adenovirus Vectors, lentiviral vectors, retroviral vectors, herpesvirus vectors, SV40 vectors, poxvirus vectors, and combinations thereof.
  • AAV adeno-associated virus
  • rAAV recombinant adeno-associated virus vector
  • scAAV self-complementing adeno-associated virus vector
  • adenovirus Vectors lentiviral vectors, retroviral vectors, herpesvirus vectors, SV40 vectors, poxvirus vectors, and combinations thereof.
  • the expression vector is an AAV vector or rAAV.
  • the composition is topically applied to at least one of: i) glial cells in the retina; ii) glial cells in the striatum, preferably in the putamen; iii ) Glial cells in the substantia nigra; iv) Glial cells in the inner ear; v) Glial cells in the spinal cord; vi) Glial cells in the prefrontal cortex; vii) Glial cells in the motor cortex; viii) Thalamus glial cells in; ix) glial cells in the ventral tegmental area (VTA); x) glial cells in the hippocampus; xi) glial cells in the cerebellum; and xii) glial cells in the brainstem Glial cells.
  • said composition further comprises i) one or more neuron-related factors, or ii) for expressing one or more neuron-related factors in said glial cells at least one expression vector.
  • the one or more neuron-related factors are selected from: AscL1, Mytl1, Ngn1, Ngn2, NeuroD1, NeuroD2, NeuroG1, Pax6, Ptbp1, P53, Zicl, Ctds1, miR- 9.miR-9-9*,miR-124,miR-124-124*,Let-7,Let-7b,miR-132,NeuroG2,Brn2,NeuroD4,Insm1,Prox1,FoxG1,Lhx6,Bcl2,Dlx1, Dlx2, Tlx3, Gata2, Gata3, Sox11, Lhx3, IsL1, etc.
  • said pharmaceutical composition or kit or kit further comprises i) one or more dopamine neuron-associated factors, or ii) for expressing in said glial cells a or at least one expression vector of multiple dopamine neuron-related factors.
  • the composition is further formulated for cell transfection, cell infection, endocytosis, Injection, intracranial administration, spinal administration, intraocular administration, intraaural administration, inhalation, parenteral administration, intravenous administration, intramuscular administration, intradermal administration, topical administration or oral administration, and ex vivo induction Transdifferentiate or reprogram and transplant the transdifferentiated or reprogrammed cells back into the body.
  • the AAV vector contains:
  • the promoter is a broad spectrum promoter or a specific promoter.
  • the broad-spectrum promoter is selected from the group consisting of CMV, CBH, CAG, PGK, SV40, EFlA, EFS, and pGlobin promoters.
  • the specific promoter is a glial cell-specific promoter or a Müller glial (MG) cell-specific promoter.
  • the glial cell-specific promoter is selected from the group consisting of GFAP promoter, ALDH1L1 promoter, EAAT1/GLAST promoter, glutamine synthetase promoter, S100 ⁇ promoter and EAAT2/GLT- 1 promoter, NG2 promoter, CD68 promoter, F4/80 promoter.
  • the MG cell-specific promoter is selected from the group consisting of GFAP promoter, ALDH1L1 promoter, Glast (also known as Slcla3) promoter and Rlbp1 promoter.
  • the transdifferentiation efficiency of glial cells is at least 0.1%, or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26% ,27%,28%,29%,30%,31%,32%,33%,34%,35%,36%,37%,38%,39%,40%,41%,42%,43 %, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or higher.
  • the disease associated with neuronal function loss or death is selected from: Parkinson's disease, schizophrenia, depression, vision impairment caused by RGC cell death, diabetes-related retinopathy, glaucoma, age Related RGC lesions, optic nerve damage, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, photoreceptor cell degeneration or death caused by injury or degeneration, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, genetics Sexual blindness, congenital amaurosis, or combinations thereof.
  • Parkinson's disease schizophrenia, depression, vision impairment caused by RGC cell death, diabetes-related retinopathy, glaucoma, age Related RGC lesions, optic nerve damage, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, photoreceptor cell degeneration or death caused by injury or degeneration, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, genetics
  • the RGCs can be integrated into the visual pathway and improve visual function.
  • the RGC can achieve functional projection to the central visual area and improve visual function.
  • said improving visual function is improving visual function in a mammal suffering from a retinal disease caused by neurodegeneration.
  • the MG cells are transdifferentiated into RGC cells, they are also differentiated into axonless cells.
  • a plurality of glial cells in the striatum are reprogrammed or transdifferentiated, and at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23% , 24%, 25%, 26%, 27%, 28%, 29%, or at least 30% of glial cells were converted into dopamine neurons.
  • the mammal includes a mammal suffering from a disease associated with loss of neuronal function or death.
  • the mammal includes a human or non-human mammal.
  • the non-human mammal includes rodents (such as mice, rats, or rabbits), primates (such as monkeys).
  • the gene editor is driven by a glial cell-specific promoter (eg, the GFAP promoter).
  • a glial cell-specific promoter eg, the GFAP promoter
  • the gene editor includes one or more gRNA and gene editing proteins.
  • the gRNA guides the gene editing protein to specifically bind to the RNA of the gene.
  • the gRNA guides the gene editing protein to specifically bind to the mRNA of the gene.
  • nucleotide sequence of the gRNA is shown in Table 1 below.
  • u is used interchangeably with the letter “t” in the context of RNA sequences to represent uridine or uridylic acid.
  • the source of the gene editing protein is selected from: Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium, Ruminococcus flavefaciens, or combinations thereof.
  • the gene or its RNA or its encoded protein is derived from a mammal; preferably, derived from a human, monkey, mouse, rat, or rabbit; more preferably, derived from a human.
  • the genes include wild-type genes and mutant genes.
  • the mutant type includes a mutant form in which the function of the encoded protein is not changed after mutation (ie, the function is the same or substantially the same as that of the wild-type encoded protein).
  • polypeptide encoded by the mutant gene is the same or substantially the same as the polypeptide encoded by the wild-type gene.
  • the mutant gene includes a homology of ⁇ 80% (preferably ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93% or ⁇ 94%, more Preferably ⁇ 95%, ⁇ 96% or ⁇ 97%, more preferably ⁇ 98% or 99%) polynucleotides.
  • the mutant gene includes truncating or adding 1-60 (preferably 1-30, more preferably 1-10) at the 5' end and/or 3' end of the wild-type gene. ) nucleotide polynucleotide.
  • the gene includes a cDNA sequence, a genomic sequence, or a combination thereof.
  • the protein includes active fragments or derivatives thereof.
  • the homology of the active fragment or derivative thereof to the gene or its RNA or its encoded protein is at least 90%, 91%, 92%, 93% or 94%, preferably 95%, 96% or 97%, more preferably 98%, 99%.
  • the active fragment or derivative thereof has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity.
  • amino acid sequence of the protein is selected from:
  • nucleotide sequence of said gene is selected from:
  • nucleotide sequence and SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 The homology of the nucleotide sequences shown in 40, 42, 44, 46, 48 and 50 is ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93% or ⁇ 94% (preferably ⁇ 95%, ⁇ 96% or ⁇ 97%, more preferably ⁇ 98% or 99%) of the polynucleotide;
  • the protein is as shown in the above amino acid sequence.
  • nucleic acid encoding the protein is as shown in the above nucleotide sequence.
  • the region targeted by the inhibitor is positions 121931-121963 (RCOR1), 2364-2393 (RCOR2), and 53556-53585 of the gene sequence. bits (RCOR3), bits 59338-59367 (Sin3a), bits 12378-12406 (Sin3b), bits 24648-24678 (HDAC1), and bits 60126-60154 (KDM1A).
  • the inhibitor or enhancer inhibits or enhances the activity and/or expression of the gene or its RNA or its encoded protein.
  • the concentration of the inhibitor or enhancer is >1 ⁇ 10 12 .
  • the inhibition rate or enhancement rate of the inhibitor or enhancer on the activity and/or expression of the gene or its RNA or its encoded protein is greater than 90%, preferably, 90%-95 %.
  • the inhibitor or enhancer targets astrocytes of brain tissue.
  • the inhibitor or enhancer targets MG cells of the retina.
  • the gRNA guides the gene editing protein to specifically bind to the mRNA of the gene.
  • the composition includes a pharmaceutical composition.
  • the composition also includes other drugs for preventing and/or treating diseases associated with loss of neuronal function or death.
  • the composition further includes other drugs for the treatment of neurological diseases associated with neuronal death.
  • the composition also includes other drugs for the prevention and/or treatment of retinal diseases.
  • the expression vector of the gene editing protein includes a vector targeting glial cells.
  • the expression vector of the gene editing protein includes a vector targeting astrocytes in brain tissue.
  • the expression vector of the gene editing protein includes a vector targeting retinal MG cells.
  • the vector includes AAV2 or AAV9.
  • the gene encoding the gene editing protein and the gRNA are located in the same expression vector (such as an AAV vector).
  • the expression vector of the gene editing protein and the expression vector of gRNA are the same expression vector (such as AAV vector).
  • the expression vector further includes a glial cell-specific promoter (eg, GFAP promoter) for driving the expression of the gene editing protein.
  • a glial cell-specific promoter eg, GFAP promoter
  • the dosage form of the composition is selected from: lyophilized formulations, liquid formulations, or combinations thereof.
  • the dosage form of the composition is a liquid formulation.
  • the dosage form of the composition is an injectable dosage form.
  • other drugs for preventing and/or treating diseases associated with neuronal function loss or death are selected from: dopamine prodrugs, non-ergot dopamine receptor agonists, monoamine oxidase B inhibitors, or their combination.
  • the composition is a cellular preparation.
  • the expression vector of the gene editing protein and the expression vector of the gRNA are the same vector or different vectors.
  • the weight ratio of component (a) to component (b) is 100:1-0.01:1, preferably, 10:1-0.1:1, more preferably, 2: 1-0.5:1.
  • the content of component (a) in the composition is 0.001%-99%, preferably 0.1%-90%, more preferably 1%-70%.
  • the content of component (b) in the composition is 0.001%-99%, preferably, 0.1%-90%, more preferably, 1%-70%.
  • the content of component (c) in the composition is 1%-99%, preferably 10%-90%, more preferably 30%-70%.
  • the component (a) and component (b) and optional component (c) account for 0.01-99.99wt% of the total weight of the composition, Preferably 0.1-90wt%, more preferably 1-80wt%.
  • the present disclosure provides a kit comprising:
  • the kit further contains:
  • (c1) a third container, and other drugs located in said third container for preventing and/or diseases associated with neuronal loss or death, and/or containing other drugs for preventing and/or treating retinal diseases, and /or contains other drugs for the treatment of neurological diseases associated with neuronal death.
  • first container, the second container, and the third container are the same or different containers.
  • the drug in the first container is a single preparation containing a gene editing protein or its expression vector.
  • the drug in the second container is a single preparation containing gRNA or its expression vector.
  • the medicine in the third container is a single preparation containing other medicines pre-used to treat neurological diseases related to neuron death.
  • the dosage form of the drug is selected from: lyophilized preparations, liquid preparations, or combinations thereof.
  • the dosage form of the medicament is an oral dosage form or an injection dosage form.
  • the kit further contains instructions.
  • Item 1 Methods for transdifferentiating mammalian non-neuronal cells into neurons, including:
  • negative regulators that can reduce the expression of negative regulatory genes, including RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL , or HMG20B, and/or
  • a positive regulator capable of increasing the expression of positively regulated genes, including DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC,
  • An effective amount of the negative regulator or the positive regulator contacts the non-neuronal cells to induce non-neuronal activity.
  • Cells are transdifferentiated into neurons.
  • the method is an in vitro, ex vivo or in vivo method.
  • RCOR1 is the amino acid sequence shown in SEQ ID NO: 1.
  • RCOR2 is the amino acid sequence shown in SEQ ID NO:3.
  • RCOR3 is the amino acid sequence shown in SEQ ID NO: 5.
  • Sin3a is the amino acid sequence shown in SEQ ID NO:7.
  • Sin3b is the amino acid sequence shown in SEQ ID NO: 9.
  • HDAC1 is the amino acid sequence shown in SEQ ID NO: 11.
  • HDAC2 is the amino acid sequence shown in SEQ ID NO: 13.
  • KDM1A is the amino acid sequence shown in SEQ ID NO: 15.
  • PHF21A is the amino acid sequence shown in SEQ ID NO: 17.
  • BAF53a is the amino acid sequence shown in SEQ ID NO: 19.
  • G9a is the amino acid sequence shown in SEQ ID NO: 21.
  • USP14 is the amino acid sequence shown in SEQ ID NO: 23.
  • HuR is the amino acid sequence shown in SEQ ID NO: 25.
  • BRG1 is the amino acid sequence shown in SEQ ID NO: 27.
  • EZH2 is the amino acid sequence shown in SEQ ID NO: 29.
  • CDYL is the amino acid sequence shown in SEQ ID NO: 31.
  • HMG20B is the amino acid sequence shown in SEQ ID NO: 33.
  • DPYSL2 is the amino acid sequence shown in SEQ ID NO: 35.
  • BAF45b is the amino acid sequence shown in SEQ ID NO: 37.
  • SCF is the amino acid sequence shown in SEQ ID NO: 39.
  • HuB is the amino acid sequence shown in SEQ ID NO: 41.
  • HuC is the amino acid sequence shown in SEQ ID NO: 43.
  • HuD is the amino acid sequence shown in SEQ ID NO: 45.
  • CYP1B1 is the amino acid sequence shown in SEQ ID NO: 47.
  • BTRC is the amino acid sequence shown in SEQ ID NO: 49.
  • Item 2 The method according to Item 1, wherein the non-neuronal cells are derived from humans.
  • Item 3 The method according to Item 1 or Item 2, wherein the non-neuronal cells are stem cells, progenitor cells or terminally differentiated cells; preferably, they are glial cells; more preferably, the glial cells are astrocytes. Glial cells, oligodendrocytes, microglia, NG2 cells, Müller glia, glioma cells or spiral ganglion glia, more preferably, the glial cells are astrocytes Plasmoblasts or Müller glia.
  • the non-neuronal cells are stem cells, progenitor cells or terminally differentiated cells; preferably, they are glial cells; more preferably, the glial cells are astrocytes. Glial cells, oligodendrocytes, microglia, NG2 cells, Müller glia, glioma cells or spiral ganglion glia, more preferably, the glial cells are astrocytes Plasmoblasts or Müller glia.
  • Item 4 The method according to Item 3, wherein the non-neuronal cells are derived from the brain.
  • the non-neuronal cells are from the brain, midbrain, cerebellum, brainstem, and spinal cord; more preferably, Comes from the striatum or substantia nigra in the brain.
  • Item 5 The method according to Item 4, wherein the non-neuronal cells are astrocytes derived from the brain, preferably astrocytes derived from the striatum or the substantia nigra.
  • Item 6 The method according to Item 4, wherein the non-neuronal cells are derived from the eye.
  • the non-neuronal cells are Müller glial cells derived from the eye.
  • Item 7 The method according to any one of Items 1 to 6, wherein the neurons are dopaminergic neurons, retinal ganglion cells, photoreceptor cells, 5-HT neurons, NE neurons, ChAT neurons, Motor neurons, GABA neurons, glutamatergic neurons, spinal cord neurons, spinal motor neurons, spinal sensory neurons, bipolar cells, amacrine cells, cochlear nerve cells, pyramidal neurons, interneurons , medium spiny neurons, Purkinje cells, granule cells, olfactory sensory neurons, or periglomerular cells, or combinations thereof.
  • the neurons are dopaminergic neurons, retinal ganglion cells, photoreceptor cells, 5-HT neurons, NE neurons, ChAT neurons, Motor neurons, GABA neurons, glutamatergic neurons, spinal cord neurons, spinal motor neurons, spinal sensory neurons, bipolar cells, amacrine cells, cochlear nerve cells, pyramidal neurons, interneurons , medium spiny neurons, Purkinje cells, granule cells, olfactory sensory neurons, or periglomerular cells,
  • Item 8 The method according to any one of Items 1 to 7, wherein the negative regulatory gene is RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, KDM1A, BAF53a, G9a, HuR, BrG1, or EZH2, and the non- Neurons are animal cells derived from the brain or eyes.
  • the negative regulatory gene is RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, KDM1A, BAF53a, G9a, HuR, BrG1, or EZH2
  • the non- Neurons are animal cells derived from the brain or eyes.
  • Item 9 The method according to Item 8, wherein the negative regulatory gene is RCOR1, RCOR2, RCOR3 or G9a, and the non-neuronal animal cells are derived from the brain; preferably, the non-neuronal cells are derived from Astrocytes of the brain; more preferably, the non-neuronal cells are astrocytes from the striatum or substantia nigra.
  • the negative regulatory gene is RCOR1, RCOR2, RCOR3 or G9a
  • the non-neuronal animal cells are derived from the brain; preferably, the non-neuronal cells are derived from Astrocytes of the brain; more preferably, the non-neuronal cells are astrocytes from the striatum or substantia nigra.
  • Item 10 The method according to Item 8, wherein the negative regulatory gene is Sin3a, Sin3b, KDM1A, BAF53a, G9a, HuR, BrG1, CDYL, or EZH2, and the non-neuronal cells are derived from the eye; preferably , the non-neuronal cells are Müller glial cells from the eye.
  • the negative regulatory gene is Sin3a, Sin3b, KDM1A, BAF53a, G9a, HuR, BrG1, CDYL, or EZH2
  • the non-neuronal cells are derived from the eye; preferably , the non-neuronal cells are Müller glial cells from the eye.
  • Item 11 The method according to item 10, wherein the negative regulatory gene is HuR and the neuron is an optic ganglion cell.
  • Item 12 The method according to Item 10, wherein the negative regulatory gene is Sin3a, KDM1A, BAF53a, G9a, HuR, BrG1, CDYL, or EZH2, and the neuron is a photoreceptor cell.
  • Item 13 The method according to any one of Items 1 to 7, wherein the positive regulatory gene is DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC, and the non-neuronal cells are from the brain ;
  • the non-neuronal cells come from the striatum, substantia nigra, ventral tegmental area of the midbrain, spinal cord, hypothalamus, dorsal midbrain, cerebral cortex, hippocampus, cerebellum; more preferably, from the striatum body.
  • Item 14 The method according to Item 13, wherein the positive regulatory gene is SCF or HuB.
  • Item 15 The method according to Item 13, wherein the positive regulatory gene is HuB or BTRC, and an effective amount of the positive regulator is brought into contact with the non-neuronal cells to induce non-neuronal non-neuronal cells. Transdifferentiate into dopaminergic neurons.
  • Item 16 The method according to any one of Items 13 to 15, wherein the non-neuronal cells are astrocytes; more preferably, the non-neuronal cells are astrocytes from the striatum or the substantia nigra. Glial cells.
  • Item 17 The method according to any one of Items 1 to 7, wherein the positive regulatory gene is DPYSL2, BAF45b, SCF, HuC, HuD, or CYP1B1, and the non-neuronal cells are from the eye; preferably, The non-neuronal cells are Müller glial cells from the eye.
  • the positive regulatory gene is DPYSL2, BAF45b, SCF, HuC, HuD, or CYP1B1
  • the non-neuronal cells are from the eye; preferably, The non-neuronal cells are Müller glial cells from the eye.
  • Item 18 The method according to Item 17, wherein the positive regulatory gene is SCF, HuD, or CYP1B1, and the neuron is an optic ganglion cell.
  • Item 19 The method according to Item 17, wherein the positive regulatory gene is DPYSL2 or BAF45b and the neuron is a photoreceptor cell.
  • Item 20 The method according to any one of Items 1 to 19, wherein said reducing the expression of a negative regulatory gene is capable of reducing the gene level of the negative regulatory gene, or reducing the mRNA level of the negative regulatory gene, or reducing the encoding of the negative regulatory gene. protein expression level;
  • Improving the expression of positively regulated genes can increase the gene level of positively regulated genes, or increase the mRNA level of positively regulated genes, or increase the expression level of encoded proteins of positively regulated genes.
  • Item 21 The method according to item 20, wherein the negative regulator is selected from the group consisting of gene editing tools or epigenetic regulation tools that reduce the expression of negatively regulated genes; inhibitors of negatively regulated genes, inhibitors of negatively regulated gene activity , or a degradation activator of the protein encoded by a negative regulatory gene.
  • the negative regulator is selected from the group consisting of gene editing tools or epigenetic regulation tools that reduce the expression of negatively regulated genes; inhibitors of negatively regulated genes, inhibitors of negatively regulated gene activity , or a degradation activator of the protein encoded by a negative regulatory gene.
  • Item 22 The method according to Item 21, wherein the inhibitor is: an inhibitory antibody of a negatively regulated gene; or a small molecule inhibitor of a negatively regulated gene; or an inhibitory mRNA, microRNA, siRNA, or shRNA, antisense oligonucleotide, binding protein or protein domain, polypeptide, nucleic acid aptamer, or PROTAC; or inhibitory binding protein or ligand that negatively regulates genes.
  • the inhibitor is: an inhibitory antibody of a negatively regulated gene; or a small molecule inhibitor of a negatively regulated gene; or an inhibitory mRNA, microRNA, siRNA, or shRNA, antisense oligonucleotide, binding protein or protein domain, polypeptide, nucleic acid aptamer, or PROTAC; or inhibitory binding protein or ligand that negatively regulates genes.
  • Item 23 The method according to item 21, wherein the negative regulator contains the gRNA and gene editing protein described in any one of SEQ ID NO: 51-67.
  • Item 24 The method according to item 20, wherein the positive regulator is selected from the group consisting of epigenetic regulatory tools capable of increasing expression of positively regulated genes, activators of positively regulated gene expression, and degradation inhibitors of proteins encoded by positively regulated genes. , a stabilizer of positive regulatory gene mRNA, or an exogenous positive regulatory gene or a functional fragment of a positive regulatory gene.
  • Item 25 The method according to item 24, wherein the activator is: an agonistic antibody that positively regulates a gene; or a small molecule agonist that positively regulates a gene; or an agonistic binding protein or ligand that positively regulates a gene; or inhibitors of competitive genes that positively regulate genes.
  • the activator is: an agonistic antibody that positively regulates a gene; or a small molecule agonist that positively regulates a gene; or an agonistic binding protein or ligand that positively regulates a gene; or inhibitors of competitive genes that positively regulate genes.
  • Item 26 The method of item 24, wherein the positive regulator contains a nucleic acid sequence as shown in SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, or 50, or contains as SEQ ID NO: A functional fragment of the nucleic acid sequence shown in 36, 38, 40, 42, 44, 46, 48, or 50.
  • Item 27 The gene editing tool according to item 21, wherein the gene editing tool includes a gene editing system or an expression vector thereof, and the gene editing system is selected from: CRISPR system (including CRISPR/Cas system), ZFN system , TALEN system, or combination thereof.
  • CRISPR system including CRISPR/Cas system
  • ZFN system ZFN system
  • TALEN system TALEN system
  • a CRISPR system is used to reduce the expression or activity of a negative regulatory gene; preferably, the CRISPR system contains a nucleic acid encoding a Cas enzyme or a functional domain of a Cas enzyme and a nucleic acid targeting the gRNA of cell transdifferentiation factor; more preferably, the Cas enzyme is Cas13; more preferably, the Cas enzyme is Cas13d, Cas13X, Cas13a, Cas13b, Cas13c, or Cas13Y; more preferably, the Cas enzyme is CasRx .
  • Item 29 The method according to item 20, wherein the negative regulator or positive regulator is carried by a carrier; preferably, the carrier is a viral vector, lipid nanoparticle (LNP), lipid bodies, cationic polymers (such as PEI), nanoparticles, exosomes, or virus-like particles; more preferably, the carrier is an AAV vector or lipid nanoparticles.
  • the carrier is a viral vector, lipid nanoparticle (LNP), lipid bodies, cationic polymers (such as PEI), nanoparticles, exosomes, or virus-like particles; more preferably, the carrier is an AAV vector or lipid nanoparticles.
  • Item 30 The method according to any one of Items 1 to 29, wherein the effective amount of the negative regulator or the positive regulator is contacted with the non-neuronal cells in vitro to induce non-neuronal cells. Cells are transdifferentiated into neurons in vitro; or
  • the effective amount of the negative regulator or the positive regulator contacts the non-neuronal cells in vivo to induce transdifferentiation of the non-neuronal cells into neurons in vitro.
  • Item 31 Use of the negative regulator or positive regulator involved in any one of Items 1 to 30 for preparing a medicament for preventing or treating diseases related to neuron damage or neuron death.
  • Item 32 Use of the negative regulator or positive regulator involved in any one of Items 1 to 30 for preventing or treating diseases related to neuron damage or neuron death.
  • Item 33 The use according to Item 31 or 32, wherein the drug is formulated as a pharmaceutical agent for administration to the nervous system, visual system and auditory system in vivo, for example, administration to the striatum, substantia nigra, midbrain in vivo Ventral tegmental area, spinal cord, hypothalamus, dorsal midbrain, cerebral cortex, hippocampus, cerebellum, subretinal, vitreous cavity, inner ear cochlea and vestibule, preferably striatum, substantia nigra, subretinal and vitreous cavity.
  • Item 34 The use according to Item 31 or 32, wherein the disease related to neuron damage or neuron death is selected from the group consisting of Parkinson's disease, visual system diseases related to RGC or photoreceptor cell function loss or death, and Alzheimer's disease.
  • Alzheimer's disease brain injury, Huntington's disease, epilepsy, depression, sleep disorders, cerebral ischemia, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, ataxia, PloyQ disease, schizophrenia disease, addiction, Pick's disease, blindness, and deafness; preferably, it is Parkinson's disease and visual system diseases related to RGC or photoreceptor cell function loss or death.
  • Item 35 The use according to Item 34, wherein the visual system disease related to RGC or photoreceptor cell function loss or death is preferably from the group consisting of: visual impairment caused by RGC cell or photoreceptor cell death, glaucoma, and age-related RGC lesions.
  • the visual system disease related to photoreceptor cell function loss or death is preferably from : Photoreceptor cell degeneration or death caused by injury or degenerative disease, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, hereditary blindness, congenital amaurosis, or combinations thereof.
  • Item 36 The use according to Item 31 or 32, wherein the neurons are dopaminergic neurons, 5-HT neurons, NE neurons, ChAT neurons, GABA neurons, glutamatergic neurons, Motor neurons, photoreceptor cells (such as rods and cones), retinal ganglion cells (RGC), cochlear nerve cells (such as cochlear spiral ganglion cells and vestibular neurons), or medium spiny neurons (MSN) or combinations thereof, preferably dopaminergic neurons, retinal ganglion cells and photoreceptor cells.
  • the neurons are dopaminergic neurons, 5-HT neurons, NE neurons, ChAT neurons, GABA neurons, glutamatergic neurons, Motor neurons, photoreceptor cells (such as rods and cones), retinal ganglion cells (RGC), cochlear nerve cells (such as cochlear spiral ganglion cells and vestibular neurons), or medium spiny neurons (MSN) or combinations thereof, preferably dopaminergic neurons, retinal ganglion cells and photoreceptor cells.
  • Item 37 A method for preventing or treating diseases related to neuron damage or neuron death, comprising giving an effective amount of the negative regulator or positive regulator involved in any one of items 1 to 30 to a subject in need. Conditioner.
  • Item 38 The method according to Item 37, wherein the disease related to neuron damage or neuron death is selected from the group consisting of Parkinson's disease, visual system diseases related to RGC or photoreceptor cell function loss or death, Alzheimer's disease, Brain injury, Huntington's disease, epilepsy, depression, sleep disorders, cerebral ischemia, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, ataxia, PloyQ disease, schizophrenia, addiction , Pick's disease, blindness, deafness; preferably Parkinson's disease and visual system diseases related to RGC or photoreceptor cell function loss or death.
  • Parkinson's disease visual system diseases related to RGC or photoreceptor cell function loss or death
  • Alzheimer's disease Brain injury, Huntington's disease, epilepsy, depression, sleep disorders, cerebral ischemia, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, ataxia, PloyQ disease, schizophrenia, addiction , Pick's disease, blindness, de
  • Item 39 The method according to Item 38, wherein the visual system disease related to RGC function loss or death is preferably From: Vision impairment due to RGC cell death, glaucoma, age-related RGC pathology, optic nerve damage, age-related macular degeneration (AMD), diabetes-related retinopathy, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, or combinations thereof ;
  • the visual system diseases related to photoreceptor cell function loss or death are preferably from: photoreceptor cell degeneration or death caused by damage or degenerative disease, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, and hereditary blindness. , congenital amaurosis, or combinations thereof.
  • Item 40 The method according to Item 37, wherein the neurons are dopaminergic neurons, 5-HT neurons, NE neurons, ChAT neurons, GABA neurons, glutamatergic neurons, motor neurons cells, photoreceptor cells (such as rods and cones), retinal ganglion cells (RGC), cochlear nerve cells (such as cochlear spiral ganglion cells and vestibular neurons), or medium spiny neurons (MSN) or other
  • the neurons are dopaminergic neurons, 5-HT neurons, NE neurons, ChAT neurons, GABA neurons, glutamatergic neurons, motor neurons cells, photoreceptor cells (such as rods and cones), retinal ganglion cells (RGC), cochlear nerve cells (such as cochlear spiral ganglion cells and vestibular neurons), or medium spiny neurons (MSN) or other
  • dopaminergic neurons, retinal ganglion cells and photoreceptor cells are preferred.
  • the disease associated with optic neuron damage or optic neuron death may be retinitis pigmentosa (RP).
  • RP retinitis pigmentosa
  • Figure 1 Results of CasRx specifically knocking down each target gene in vitro.
  • expressing CasRx and gRNA targeting RCOR1, Sin3a, HDAC2, PHF21A, BAF53a, KDM1A, G9a, USP14, HuR, BrG1, EZH2, CDYL, and HMG20B can achieve RCOR1, Sin3a, HDAC2, KDM1A, Efficient knockdown of PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, and HMG20B.
  • Figure 2 Schematic diagram of transdifferentiating mouse astrocytes into neurons by knocking down or overexpressing different target genes.
  • Vector 1 AAV-GFAP-mCherry
  • Figure 2A drives the expression of fluorescent protein mCherry by the astrocyte-specific promoter GFAP
  • vector 2 AAV-GFAP-CasRx is driven by the astrocyte-specific promoter GFAP.
  • vector 3 encodes CasRx and gRNA targeting RCOR1, Sin3a, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, or HMG20B ;
  • Vector 1 in Figure 2B is a schematic diagram of the vector for GFAP-driven mCherry
  • vector 4 is a schematic diagram of the vector for GFAP-driven expression of target genes.
  • the target genes are DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC respectively.
  • Figure 3 Conversion of astrocytes into neurons after knocking down different negative regulatory genes in the mouse striatum.
  • Figure 3A shows that mice were injected with control group AAV (AAV-GFAP-mCherry+AAV-GFAP- After CasRx), AAV-GFAP-mCherry can specifically label astrocytes without transdifferentiation into neurons.
  • the yellow arrow (the arrow in Figure 3A is a yellow arrow) indicates the astrocytes labeled by GFAP-mCherry.
  • Figure 3B to Figure 3M respectively show the use of CasRx to knock down RCOR1, HDAC2, PHF21A, BAF53a, G9a, USP14, HuR, The results of transdifferentiation of astrocytes into neurons by BrG1, EZH2, CDYL, HMG20B, and KDM1A.
  • the red signal is the cells labeled by AAV-GFAP-mCherry (the leftmost column of Figure 3A to Figure 3M is the result of mCherry labeling.
  • the bright part in the figure is red
  • NeuN is a neuron-specific marker
  • the white arrow indicates cells where the red mCherry signal is co-labeled with NeuN (the arrows in Figure 3B to Figure 3M are white arrows).
  • Scale bar is 50 microns.
  • Figure 4 Statistical diagram of astrocytes converted into neurons after knocking down different negative regulatory genes.
  • Figure 5 Overexpression of different positive regulatory genes converts astrocytes into neurons in the mouse striatum.
  • Figure 5A shows a control group AAV (AAV-GFAP-mCherry) specifically labeled astrocytes injected into the striatum of the mouse brain.
  • the yellow arrow indicates the astrocytes labeled by GFAP-mCherry.
  • Astrocytes are not co-labeled with NeuN, which is a neuron-specific marker;
  • Figure 5B to Figure 5I show the test group AAV (AAV-GFAP-mCherry+AAV-GFAP-positive) injected into the striatum of the mouse brain.
  • the results of regulatory genes among which the positively regulated genes are DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC, and the white arrows indicate cells where the red mCherry signal is co-labeled with NeuN (the leftmost column of Figure 5A to Figure 5I is the result of mCherry labeling, the bright part in the figure is red, the arrows in Figure 5B to Figure 5I are white arrows), and NeuN is a neuron-specific marker. Scale bar is 50 microns.
  • Figure 6 Statistics of astrocytes converted into neurons after up-regulation of different positive regulatory genes.
  • Figure 7 Conversion of astrocytes into dopaminergic neurons after overexpression of BTRC or HuB in mouse striatum.
  • Figure 7A shows a control group AAV (AAV-GFAP-mCherry) injected into the striatum of mice.
  • the yellow arrow indicates that the labeled mCherry-positive cells do not co-label with TH, nor do they co-label with TH.
  • TH is a dopaminergic neuron-specific protein marker
  • NeuN is a neuron-specific marker
  • Figure 7B and Figure 7C respectively show the test group AAV (AAV-GFAP- mCherry+AAV-GFAP-positive regulatory genes), in which the positive regulatory genes are HuB and BTRC, and the white arrows (the arrows in Figure 7B and C are white arrows) indicate cells where the red mCherry signal is co-labeled with NeuN and TH.
  • Scale bar is 50 microns. Among them, the bright part in the mCherry column is red, the bright part in the TH column is green, the bright part in the NeuN column is white, and the bright part in the DAPI column is blue.
  • Figure 8 Schematic diagram of converting Müller glia cells into retinal ganglion cells or photoreceptor cells by overexpressing or knocking down the target gene in the retina.
  • Vector 1 in Figure 8A is a schematic diagram of the plasmid of AAV-GFAP-EGFP-2A-Cre. Cre expression is driven by the astrocyte-specific promoter GFAP.
  • Vector 2 is a schematic diagram of the plasmid of AAV-GFAP-CasRx, which is driven by astrocyte-specific promoter GFAP. Plasma cell-specific promoter GFAP drives the expression of RNA editing protein CasRx.
  • Vector 3 is a schematic diagram of the plasmid of AAV-GFAP-CasRx-gRNA, encoding CasRx and targeted negative regulatory genes (Sin3a, HDAC2, KDM1A, BAF53a, G9a, HuR, BrG1 , EZH2, CDYL) gRNA.
  • Figure 8B shows that overexpression of each positive regulatory gene in the retina transformed Müller glia cells. Schematic diagram of differentiation into retinal ganglion cells or photoreceptor cells.
  • Vector 1 is GFAP-driven mCherry
  • vector 4 is GFAP-driven expression of the target gene.
  • the target gene is DPYSL2, BAF45b, SCF, CYP1B1, or BTRC.
  • Figure 9 Knocking down negative regulatory genes in mouse eyes converts Müller glia into photoreceptor cells.
  • Figure 9A shows AAV (AAV-GFAP-EGFP-P2A-Cre) in the control group injected into the retina of Ai9 mice to specifically label Müller glia cells.
  • the yellow arrow indicates the Müller glial cells specifically labeled by GFAP-EGFP-P2A-Cre.
  • the Müller glial cell bodies are located in the INL layer, and no tdTomato-labeled cells were found in the ONL layer. red blood cells.
  • Figure 9B to Figure 9J respectively show the subretinal injection of different AAVs (AAV-GFAP-mCherry+AAV-GFAP-CasRx-gRNA) in the test group of mice, in which the gRNA targets Sin3a, HDAC2, BAF53a, G9a, HuR, and BrG1 respectively.
  • Figure 10A and Figure 10B respectively show the transformation of Müller glial cells into photoreceptor cells after subretinal injection of test group AAV (AAV-GFAP-EGFP-P2A-Cre+AAV-GFAP-positive regulatory gene) in mice.
  • the positive regulatory genes are DPYSL2 and BAF45b respectively
  • the white arrows indicate tdTomato-positive cells in the ONL layer
  • the yellow arrows indicate tdTomato-positive Müller glia cells in the INL layer.
  • the scale bar is 50 microns.
  • the leftmost column in the figure is the result of mCherry labeling. The bright part in the left column is red.
  • the arrow in the INL column in the figure is a yellow arrow
  • the arrow in the ONL column is a white arrow.
  • Figure 11 Cytogram of Müller glia reprogrammed into photoreceptor cells.
  • Figure 12 Knocking down Sin3a or KDM1A in mouse eyes converts Müller glia into rod photoreceptor cells.
  • Figure 12A shows the results of injecting AAV-GFAP-EGFP-P2A-Cre+AAV-GFAP-CasRx into the retina of Ai9 mice.
  • the white signal is staining of the rod-specific protein marker rhodopsin, and the yellow arrow (the arrow in Figure 12A (yellow arrow) indicates that tdTomato-labeled Müller glial cells are located in the INL layer, and there are no tdTomato-positive cells in the ONL layer.
  • Figure 12B and Figure 12C show the transdifferentiation results of the test group's AAV targeting Sin3a and KDM1A (AAV-GFAP-mCherry+AAV-GFAP-CasRx-gRNA) injected under the retina of Ai9 mice.
  • the white arrow indicates the tdTomato located in the ONL layer. Positive cells were co-labeled with rhodopsin. Scale bar is 50 microns.
  • the leftmost column of Figures 12A to 12C shows the results of mCherry labeling. The bright parts in the left column are red, and the arrows in Figures 12B and C are white arrows.
  • Figure 13 Knocking down Sin3a or EZH2 in mouse eyes converts Müller glia into cones.
  • Figure 13A shows the results of injecting AAV-GFAP-EGFP-P2A-Cre into the retina of Ai9 mice.
  • GFAP-EGFP-P2A-Cre can specifically label Müller glia.
  • the cell bodies of Müller glia are located in the INL layer. No red cells labeled by tdTomato were found in the ONL layer.
  • the green signal is the EGFP fluorescence expressed by GFAP-EGFP-P2A-Cre.
  • the yellow arrow (the arrow in Figure 13A is a yellow arrow) indicates the Muller's glue co-labeled with the green signal and the red signal. Plasma cells.
  • Figure 13B shows a control group AAV (AAV-GFAP-EGFP-P2A-Cre+AAV-GFAP-CasRx) injected into the retina of Ai9 mice.
  • the white signal is the cone-specific protein marker m-CAR and the cyan arrow ( Figure The arrow in 13B (cyan arrow) points to tdTomato-positive Müller glial cells, in No tdTomato-positive cells were found in the ONL layer.
  • Figure 13C and Figure 13D are the results of subretinal injection of test group AAV (AAV-GFAP-mCherry+AAV-GFAP-CasRx-gRNA) targeting Sin3a and EZH2 in mice, respectively.
  • white arrows in Figure 13C and Figure 13D Arrows (white arrows) indicate cells that are tdTomato-positive and co-labeled with mCAR in the ONL layer. Scale bar is 50 microns.
  • the bright part in the mCherry column is red
  • the bright part in the EGFP column is green
  • the bright part in the DAPI column is blue.
  • Figure 14 Knockdown of negative regulatory genes in mouse eyes converts Müller glia cells into retinal ganglion cells.
  • Figure 14A shows the results of injecting control group AAV (AAV-GFAP-EGFP-P2A-Cre) into the retina of Ai9 mice. There was almost no labeled optic nerve in the optic nerve.
  • Figures 14B to 14I respectively show the results of subretinal injection of test group AAV (AAV-GFAP-mCherry+AAV-GFAP-CasRx-gRNA) in Ai9 mice to inhibit different negative regulatory genes, in which the gRNA targets HDAC2, BAF53a, and G9a respectively.
  • HuR, BrG1, EZH2, CDYL, KDM1A HuR, BrG1, EZH2, CDYL, KDM1A.
  • Figure 15 Figures 15A to 15G show the transformation of Müller glial cells into retinal ganglion cells by overexpressing different positive regulatory genes in mouse eyes.
  • the positively regulated genes are DPYSL2, BAF45b, SCF, HuB, HuD, CYP1B1, and BTRC, and the scale bar is 150 ⁇ m.
  • Figure 16 Statistical diagram of Müller glial cells reprogramming into retinal ganglion cells.
  • Figure 17 Results of reprogramming Müller glia into photoreceptor cells in retinitis pigmentosa disease model mice.
  • Figure 17A is a schematic diagram of the degeneration and death of rod and cone photoreceptor cells and the time points of AAV injection in the retinitis pigmentosa disease model mouse (Pde6b-KO). AAV injection was performed after all photoreceptor cells in the retina of Pde6b-KO mice died (P50), and retinal tissue was harvested for analysis 50 days after injection;
  • Figure 17B and Figure 17C are retinal harvests from Pde6b-KO mice at 4 weeks and 6 weeks respectively. tissue and immunofluorescent staining with the rod-specific marker Rhodopsin, no rods were observed at either 4 or 6 weeks.
  • Figure 17D shows the results of immunofluorescence staining of retinal tissue sections injected with AAV (GFAP-CasRx+CBH-Pde6b) in the control group. There are no Rhodopsin-positive cells in the ONL layer.
  • Figure 17E to Figure 17F show the results of immunofluorescence staining of retinal tissue sections of AAV (GFAP-CasRx-gRNA+CBH-Pde6b) injection treatment group.
  • gRNA targeted knockdown of the expression of EZH2 or KDM1A, and Rhodopsin-positive cells reappeared in the ONL layer; where , the bright color in the Rhodopsin column is green, and the bright color in the DAPI column is blue.
  • Figure 17G is a statistical diagram of the number of retinal photoreceptor cells in different treatment groups of Pde6b-KO mice.
  • 4w and 6w are the number of photoreceptor cells in the retina of Pde6b-KO mice without AAV injection.
  • the number of photoreceptor cells in the EZH2 or KDM1A knockdown group increased significantly, and
  • Figure 17H shows the thickness of the retinal ONL layer in different treatment groups of Pde6b-KO mice.
  • 4w and 6w show the thickness of the retinal ONL layer of Pde6b-KO mice without AAV injection.
  • the EZH2 or KDM1A knockdown group was significantly thicker.
  • the inventor unexpectedly discovered for the first time that the expression or activity of one or more genes or RNAs or proteins encoding them selected from the group consisting of: Expression or activity of proteins: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, and/or genes or RNAs that enhance the expression or activity of one or more genes or RNAs or proteins encoding the proteins in glial cells selected from the group consisting of: The expression or activity of its encoded proteins: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC, can effectively induce the differentiation of glial cells into neuronal cells, thereby treating neurological diseases related to neuronal function loss or death. . On this basis, the inventor completed the technical solution of the present application.
  • retinal ganglion cell (RGC) degeneration is the primary cause of permanent blindness.
  • the transdifferentiation of Müller glial cells (MG) into RGC can help restore vision.
  • MGs can be converted directly into RGCs by knocking down (e.g., knocking down using the RNA-targeted CRISPR system CasRx) the gene or its RNA or its encoded protein in mature mouse retinas.
  • NMDA N-methyl-D-aspartate
  • RGCs converted from MG achieved functional projections to central visual areas and resulted in improved visual function. . Therefore, reducing negative regulatory genes (e.g., CasRx-mediated knockdown) would be a promising therapy for the treatment of retinal diseases caused by neurodegeneration.
  • This application uses the recently characterized RNA-targeting CRISPR system CasRx to inhibit the gene or its RNA or its encoded protein.
  • Müller glia are the main glial cells in retinal tissue
  • retinal ganglion cells are nerve cells located in the innermost layer of the retina, and their dendrites mainly communicate with bipolar cells. , its axons extend to the optic nerve head to form the optic nerve.
  • differentiation has the same meaning herein and may refer to the generation of cells of a specific lineage from different types of non-neuronal cells, such as astrocytes (such as neuronal cells) without intermediate differentiation processes.
  • a negative regulator or a positive regulator is brought into contact with the non-neuronal cell
  • a negative regulator or a positive regulator is brought into contact with the non-neuronal cell
  • the positive modulator e.g., compound, nucleic acid, viral vector, etc.
  • contacting is by adding a negative regulator or a positive regulator to the cell culture.
  • Exposure may also be by injecting a negative or positive modulator, or a vehicle containing a negative or positive modulator, into a location in the body; This is accomplished by delivering the carrier of the positive regulator or positive regulator to a location in the body such that the negative regulator or positive regulator "contacts" the targeted cell type.
  • the effective amount of the negative regulator or the positive regulator is contacted with the non-neuronal cells in vivo to induce transdifferentiation of the non-neuronal cells into neurons in vitro.
  • Positive modulators or negative modulators are formulated for in vivo administration to the nervous system, visual system, and auditory system, for example, to the striatum, substantia nigra, ventral tegmental area of the midbrain, spinal cord, Hypothalamus, dorsal midbrain, cerebral cortex, hippocampus, cerebellum, subretinal, vitreous cavity, inner ear cochlea and vestibule, preferably striatum, substantia nigra, subretinal and vitreous cavity.
  • the effective amount of the negative regulator or the positive regulator is contacted with the non-neuronal cells in vitro to induce transdifferentiation of the non-neuronal cells into neurons in vitro.
  • neurons obtained through in vitro contact can be configured into appropriate cell therapy agents and administered to individuals in need through appropriate administration methods, such as intravenous infusion, in situ injection, etc.
  • NNL refers to the outer granule cell layer
  • INL refers to the inner granule cell layer
  • GCL refers to the retinal ganglion cell layer
  • non-neuronal cell may refer to any type of cell that is not a neuron.
  • the non-neuronal cells are stem cells, progenitor cells, or terminally differentiated cells.
  • non-neuronal cells are cells of cell lineages other than the neuronal lineage (eg, hematopoietic lineage).
  • the non-neuronal cells are cells of the neuronal lineage but are not neurons, such as glial cells.
  • the non-neuronal cells are non-neuronal terminally differentiated cells such as, but not limited to, glial cells, fibroblasts, embryonic fibroblasts, epithelial cells, melanocytes, keratinocytes, adipocytes , blood cells, bone marrow stromal cells, Langerhans cells, muscle cells, rectal cells or chondrocytes.
  • the non-neuronal cells are from a non-neuronal cell line, such as, but not limited to, glioblastoma cell line, HeLa cell line, NT2 cell line, ARPE19 cell line, or N2A cell line.
  • glioblastoma cell line such as, but not limited to, glioblastoma cell line, HeLa cell line, NT2 cell line, ARPE19 cell line, or N2A cell line.
  • Cell lineage or “lineage” may mean the developmental history of a tissue or organ from a fertilized embryo.
  • Neurogenesis may refer to the developmental history from neural stem cells to mature neurons, including various stages along this process (called neurogenesis), such as, but not limited to, neural stem cells (neuroepithelial cells, radial glia) , neural progenitor cells (e.g., interneuron precursors), neurons, astrocytes, oligodendrocytes, and microglia.
  • neural stem cells neuroepithelial cells, radial glia
  • neural progenitor cells e.g., interneuron precursors
  • neurons e.g., astrocytes, oligodendrocytes, and microglia.
  • the non-neuronal cells are stem cells, such as embryonic stem cells, neural stem cells, or induced pluripotent stem cells.
  • the non-neuronal cells are progenitor cells, such as neural grandmother cells or neural precursor cells (eg, dopamine neural precursor cells).
  • the non-neuronal cells are derived from the brain.
  • the non-neuronal cells are derived from the brain, midbrain, cerebellum, brainstem, and spinal cord; more preferably, they are derived from striae in the brain. body or substantia nigra.
  • the non-neuronal cells are astrocytes derived from the brain, preferably, astrocytes derived from the striatum or substantia nigra.
  • the non-neuronal cells are derived from the eye.
  • the non-neuronal cells are Müller glial cells from the eye.
  • progenitor cell refers to intermediate cells that exist in adult tissues before cells differentiate into terminally differentiated cells. The differentiation of progenitor cells is usually clear.
  • terminal differentiated cells also known as terminal cells, refers to cells of a specific type with specific functional nuclei that no longer undergo differentiation, division and proliferation. For example: glial cells, somatic cells, fibroblasts, red blood cells, mature epidermal cells, muscle cells, etc.
  • stem cells should be understood as undifferentiated cells that have differentiation potential and proliferation capacity (in particular self-renewal capacity) but retain differentiation potential. According to differentiation potential, stem cells include subpopulations such as pluripotent stem cells (PSC), multipotent stem cells, unipotent stem cells, embryonic stem cells, etc. In some implementations, the stem cells can be embryonic stem cells, neural stem cells, or induced pluripotent stem cells.
  • PSC pluripotent stem cells
  • multipotent stem cells multipotent stem cells
  • unipotent stem cells unipotent stem cells
  • embryonic stem cells etc.
  • the stem cells can be embryonic stem cells, neural stem cells, or induced pluripotent stem cells.
  • pluripotent stem cells refers to stem cells that can be cultured in vitro and have the ability to differentiate into any cell lineage belonging to the three germ layers (ectoderm, mesoderm, endoderm). PSCs can be induced from fertilized eggs, cloned embryos, germline stem cells, stem cells in tissues, somatic cells, etc. Examples of PSCs include embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs or ips), embryonic germ cells (EG cells), and the like.
  • ESCs embryonic stem cells
  • iPSCs or ips induced pluripotent stem cells
  • EG cells embryonic germ cells
  • induced pluripotent stem cells can be generated directly from adult cells by reprogramming.
  • Adult cells can be converted into PSCs by introducing the products of a specific set of pluripotency-related genes.
  • neural precursor cells refers to cells that have neuronal development potential and are in a precursor state of neuronal development.
  • neuron may have its general meaning as would be understood by those skilled in the art.
  • a neuron may refer to an electrically excitable cell that can receive, process, and transmit information through electrical signals (eg, membrane potential discharge) and chemical signals (eg, synaptic transmission of neurotransmitters).
  • electrical signals eg, membrane potential discharge
  • chemical signals eg, synaptic transmission of neurotransmitters.
  • chemical signals transduced between neurons can occur through specialized connections called synapses.
  • the neurons are dopaminergic neurons, retinal ganglion cells, photoreceptor cells, 5-HT neurons, NE neurons, ChAT neurons, motor neurons, GABA neurons, glutamate neurons Neurons, spinal neurons, spinal motor neurons, spinal sensory neurons, bipolar cells, amacrine cells, cochlear nerve cells, pyramidal neurons, interneurons, medium spiny neurons, Purkinje cells , granule cells, olfactory sensory neurons, or periglomerular cells, or combinations thereof.
  • protein As used herein, the terms “protein”, “peptide” and “polypeptide” may refer to an amino acid polymer or a group of two or more interacting or combined amino acid polymers, and have the same meaning.
  • nucleic acid and “polynucleotide” have the same meaning and may refer to a nucleic acid molecule containing one or more nucleotides.
  • target gene refers to a gene regulated by a "negative regulator” or a "positive regulator”.
  • photoreceptor refers to a type of neuroepithelial cell in the retina that is responsible for visual light transmission, including rods and cones. Some retinitis pigmentosa, macular degeneration, diabetes complications, etc. can cause the death of photoreceptor cells.
  • ganglion cells are neurons located in the final segment of the retina. Many eye diseases can lead to the death of optic ganglion cells, such as ischemic retinopathy, glaucoma, etc. The death of optic ganglion cells may also lead to permanent blindness.
  • Müller glia are the main glial cells in retinal tissue
  • retinal ganglion cells are nerve cells located in the innermost layer of the retina. Their dendrites mainly communicate with bipolar cells. , its axons extend to the optic nerve head to form the optic nerve.
  • vector refers to a tool that can transport exogenous nucleic acids into cells, which can be nucleic acids, proteins, etc.
  • the vector is capable of directing the synthesis of one or more proteins encoded by one or more genes carried by the vector or microRNA encoded by a polynucleotide carried by the vector.
  • vectors can be used to introduce the polynucleotides provided herein. In this article, the vector can be a variety of different forms of vectors.
  • the vector can be a viral vector, a plasmid vector, a minicircle vector, a linear DNA vector, a doggybone vector, a lipid vector, or a vector. Plastids, nanoparticles, exosomes, extracellular vesicles, cationic polymers (such as PEI) or virus-like particles, etc.
  • viral vector is a nucleic acid of viral origin that may be capable of transporting another nucleic acid into a cell. When a viral vector is present in an appropriate environment, it is capable of directing the expression of one or more proteins encoded by one or more genes carried by the vector or microRNA encoded by a polynucleotide carried by the vector.
  • viral vectors include, but are not limited to, retroviral vectors, adenoviral vectors, lentiviral vectors, poxviral vectors, herpesviral vectors, and adeno-associated viral vectors.
  • nanoparticle carriers may include polymer-based nanoparticles, aminolipid-based nanoparticles, and metal nanoparticles.
  • the vectors provided herein can be used to deliver the polynucleotide compositions provided herein.
  • a single vector is used to deliver at least about 2, 3, 4, or up to 5 polynucleotides.
  • a single vector is used to deliver at least about 2, 3, 4, or up to 5 different polynucleotides.
  • a single vector is used to deliver at least about 2, 3, 4, or up to 5 identical polynucleotides.
  • the vector can deliver DNA (eg, double-stranded DNA or single-stranded DNA), and can also deliver RNA.
  • RNA can include base modifications.
  • Vectors may include recombinant vectors.
  • the vector may be a vector modified from a naturally occurring vector.
  • the carrier may include at least a portion of a non-naturally occurring carrier. Any carrier can be utilized.
  • astrocytes may refer to the star-shaped glial cells that are characteristic of the brain and spinal cord. It will be clear to those skilled in the art that astrocytes can be characterized by being star-shaped, expressing markers such as glial fibrillary acidic protein (GFAP) and aldehyde dehydrogenase 1 family member L1 (ALDH1L1), excitatory Excitatory amino acid transporter 1/glutamate aspartate transporter (EAAT1/GLAST), glutamine synthetase, S100 ⁇ or excitatory amino acid transporter 1/glutamate transporter 1 (EAAT2/GLT-1), Involved with endothelial cells in the blood-brain barrier, transmitter uptake and release, regulation of ion concentrations in the extracellular space, response to neuronal injury and involvement in nervous system repair, and metabolic support of peripheral neurons.
  • markers such as glial fibrillary acidic protein (GFAP) and aldehyde dehydrogenase 1 family member L1 (ALDH1
  • astrocytes may refer to non-neuronal cells in the nervous system that express glial fibrillary acidic protein (GFAP), aldehyde dehydrogenase 1 family member L1 (ALDH1L1), or both. .
  • astrocytes may refer to non-neuronal cells in the nervous system that express glial fibrillary acidic protein (GFAP) promoter-driven transgenes (e.g., red fluorescent protein (RFP), Cre recombinase) .
  • GFAP glial fibrillary acidic protein
  • RFP red fluorescent protein
  • glial cells useful in the methods provided herein are glial cells isolated from the brain. In some embodiments, astrocytes useful in the methods provided herein are star-shaped glial cells in the brain or spinal cord.
  • an "effective amount" of the negative modulator or the positive modulator in contact with the non-neuronal cells refers to the amount of the negative modulator or positive modulator that is capable of converting the non-neuronal cells into neurons. , or the amount of carrier containing a negative regulator or a positive regulator.
  • active fragment As used herein, “active fragment”, “functional fragment”, and “functional fragment” have the same meaning and refer to a truncated fragment of a gene or protein that has the same or similar function as the full-length gene or protein.
  • “functional fragment of SEQ ID NO: 36” refers to a truncated fragment of SEQ ID NO: 36 that has a similar function to the sequence shown in SEQ ID NO: 36. This truncated fragment can perform the same function as SEQ ID NO: 36.
  • NO: 36 has the same or similar positive regulatory effect, promoting the transdifferentiation of non-neuronal cells into neurons.
  • an inhibitor of a negatively regulated gene may be an RNA interference agent that contains RNA and mediates targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • RNA interference agents direct the sequence-specific degradation of mRNA through the process of RNA interference (RNAi).
  • RNA interference agents can inhibit the expression of one or more negatively regulated genes in cells.
  • RNA interference agents include, but are not limited to, "small interfering RNA (siRNA)", “endoribonuclease-prepared siRNA (e-siRNA)", “short hairpin RNA (shRNA)” and “small time-regulated RNA” ( stRNA), “cleaved siRNA (d-siRNA),” and aptamers, oligonucleotides, and other synthetic nucleic acids containing at least one uracil base.
  • siRNA small interfering RNA
  • e-siRNA endoribonuclease-prepared siRNA
  • shRNA short hairpin RNA
  • stRNA small time-regulated RNA
  • d-siRNA cleaved siRNA
  • aptamers oligonucleotides, and other synthetic nucleic acids containing at least one uracil base.
  • RNA interfering agents are delivered by a carrier , such vectors include, but are not limited to, replication-deficient or replication-competent viral vectors (eg, adenovirus, lentivirus, gamma retrovirus, adeno-associated virus, etc.).
  • viral vectors eg, adenovirus, lentivirus, gamma retrovirus, adeno-associated virus, etc.
  • the invention provides methods of generating neurons in vivo.
  • Exemplary methods include administering a positive or negative modulator to an area of the subject's nervous system, such as the brain, eye, or spinal cord, and allowing non-neuronal cells to reprogram into functional neurons.
  • expression vectors refers to vectors that add expression elements (such as promoters, RBS, GOI, terminators, etc.) to the basic skeleton of the cloning vector to enable the expression of the target gene. Constructing an expression vector allows the target gene to be expressed and function in recipient cells.
  • expression elements such as promoters, RBS, GOI, terminators, etc.
  • a "negatively regulated gene” refers to a gene that, when the expression of the gene, or the expression of its mRNA, or the expression of the protein encoded by the gene is reduced or inhibited, can increase, promote or improve the transition of non-neuronal cells to neurons.
  • Differentiation genes such as: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, etc.
  • a negative regulator refers to a regulator that is capable of reducing or inhibiting the gene expression, or the expression of mRNA, or the expression of the protein encoded by the "negatively regulated gene".
  • a negative regulator can be a gene editing tool or epigenetic regulation tool that can reduce the expression of a negatively regulated gene; it can also be an inhibitor of a negatively regulated gene, an inhibitor of a negatively regulated gene activity, or a negative regulator. Genes encoding proteins that are activators of degradation.
  • a "negative regulator” can be an inhibitory antibody of a negatively regulated gene; or a small molecule inhibitor of a negatively regulated gene; or an inhibitory mRNA, microRNA, siRNA, shRNA, or antisense of a negatively regulated gene. Oligonucleotides, binding proteins or protein domains, polypeptides, nucleic acid aptamers, or PROTACs; or inhibitory binding proteins or ligands that negatively regulate genes.
  • the gene editing tool used as a "negative regulator” can be a CRISPR gene editing tool, including a gene editing protein and a gRNA targeting a negative regulatory gene.
  • the gene editing protein can be a DNA editing protein, such as Cas9, or an RNA editing protein, such as Cas13.
  • the gene editing tool used as a "negative regulator” can be a gene editing tool containing a zinc finger nuclease, or it can be a TALENs (transcription activator-like (TAL) effector nucleases)) gene editing tool. .
  • TALENs transcription activator-like (TAL) effector nucleases
  • the “negative regulator” may be an epigenetic regulation tool.
  • Epigenetic regulation tools refer to the use of epigenetic modification methods (such as methylation, acetylation, phosphorylation, chromatin conformation changes, etc.) without changing the nucleotide sequence of the target gene.
  • Proteins, genes, small molecule compounds, etc. used to regulate the content and function of nucleic acids or proteins. For example: fusion of epigenetic regulatory proteins such as KRAB and DNMT3A on the mutated CRISPR/Cas system.
  • a "positively regulated gene” means that when the expression of the gene, or the expression of its mRNA, or the protein encoded by the gene When the expression of white is increased or activated, genes that can increase, promote or improve the transdifferentiation of non-neuronal cells into neurons, such as: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC.
  • a “positive regulator” refers to a regulator that is capable of increasing or activating the gene expression of a “positive regulator gene", or the expression of mRNA, or the expression of the protein encoded by the gene.
  • the positive regulator can be an epigenetic regulation tool that can improve the expression of a positively regulated gene, an activator of positively regulated gene expression, a degradation inhibitor of the protein encoded by a positively regulated gene, or a stabilizer of the mRNA of a positively regulated gene. , or exogenous positive regulatory genes or functional fragments of positive regulatory genes.
  • a "positive regulator” can be: an agonistic antibody that positively regulates a gene; or a small molecule agonist that positively regulates a gene; or an agonistic binding protein or ligand that positively regulates a gene; or a positive regulator.
  • Competitive gene inhibitors of genes can be: an agonistic antibody that positively regulates a gene; or a small molecule agonist that positively regulates a gene; or an agonistic binding protein or ligand that positively regulates a gene; or a positive regulator.
  • diseases related to neuronal function loss or neuron death may be Parkinson's disease, visual system diseases related to RGC or photoreceptor cell function loss or death, Alzheimer's disease, brain injury, Huntington's disease , Epilepsy, Depression, Sleep Disorders, Cerebral Ischemia, Motor Neurone Disease, Amyotrophic Lateral Sclerosis, Spinal Muscular Atrophy, Ataxia, PloyQ Disease, Schizophrenia, Addiction, Pick's Disease, RGC or Blindness, macular degeneration, retinitis pigmentosa, deafness, night blindness, color blindness, hereditary blindness, congenital amaurosis, etc. caused by damage or death of photoreceptor cells.
  • the diseases related to neuron function loss or neuron death are preferably Parkinson's disease and visual system diseases related to RGC or photoreceptor cell function loss or death, for example: dopamine neuron function loss or death related diseases Disease, vision impairment associated with loss or death of optic ganglia or photoreceptor cells.
  • Visual system diseases related to RGC or photoreceptor cell function loss or death are preferably from: vision impairment caused by RGC cell or photoreceptor cell death, glaucoma, age-related RGC lesions, optic nerve damage, age-related macular degeneration (AMD), diabetes-related Retinopathy, retinal ischemia or hemorrhage, Leber's hereditary optic neuropathy, or a combination thereof;
  • the visual system disease related to photoreceptor cell function loss or death is preferably from: photoreceptor cell degeneration or death caused by injury or degenerative disease, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, hereditary blindness, congenital amaurosis, or combinations thereof.
  • Astrocytes are the most abundant type of cells in the mammalian brain. They perform many functions, including biochemical support (such as forming the blood-brain barrier), providing nutrients to neurons, maintaining extracellular ion balance, and participating in repair and scarring after brain and spinal cord injury. Astrocytes can be divided into two types based on the content of glial filaments and the shape of their processes: fibrous astrocytes are mostly distributed in the white matter of the brain and spinal cord, with slender processes and fewer branches. , the cytoplasm contains a large number of glial filaments; protoplasmic astrocytes are mostly distributed in the gray matter, with thick and short cell processes and many branches.
  • Astrocytes that can be used in the present disclosure are not particularly limited and include various astrocytes derived from the mammalian central nervous system, for example, from the striatum, substantia nigra, midbrain ventral tegmental area, inferior The thalamus, spinal cord, dorsal midbrain or cerebral cortex, preferably, originate from the striatum and substantia nigra.
  • functional neurons may refer to neurons capable of sending or receiving information through chemical or electrical signals.
  • functional neurons exhibit one or more functional properties of mature neurons present in the normal nervous system, including, but not limited to: excitability (e.g., the ability to exhibit action potentials, e.g., rapid rise and subsequent fall) (voltage or membrane potential across the cell membrane), formation of synaptic connections with other neurons, presynaptic neurotransmitter release, and postsynaptic responses (e.g., excitatory postsynaptic currents or inhibitory synaptic after-touch current).
  • excitability e.g., the ability to exhibit action potentials, e.g., rapid rise and subsequent fall
  • postynaptic responses e.g., excitatory postsynaptic currents or inhibitory synaptic after-touch current.
  • functional neurons are characterized by expressing one or more markers of functional neurons, including but not limited to synaptophysin, synaptophysin, glutamate decarboxylase 67 (GAD67), glutamine Acid decarboxylase 65 (GAD65), parvalbumin, dopamine- and cAMP-regulated neuronal phosphoprotein 32 (DARPP32), vesicular glutamate transporter 1 (vGLUT1), vesicular glutamate transporter 2 (vGLUT2) , acetylcholine, tyrosine hydroxylase (TH), dopamine, vesicular GABA transporter (VGAT) and gamma-aminobutyric acid (GABA).
  • GABA glutamate decarboxylase 67
  • GAD65 glutamine Acid decarboxylase 65
  • DARPP32 dopamine- and cAMP-regulated neuronal phosphoprotein 32
  • vGLUT1 vesicular glutamate transporter 1
  • functional neurons can be dopaminergic neurons, 5-HT neurons, NE neurons, ChAT neurons, GABA neurons, glutamatergic neurons, motor neurons, photoreceptor cells (e.g. rods and cones), retinal ganglion cells (RGC), cochlear nerve cells (such as cochlear spiral ganglion cells and vestibular neurons), or medium spiny neurons (MSN) or combinations thereof, preferably dopaminergic nerves cells, retinal ganglion cells, and photoreceptor cells.
  • photoreceptor cells e.g. rods and cones
  • RRC retinal ganglion cells
  • cochlear nerve cells such as cochlear spiral ganglion cells and vestibular neurons
  • MSN medium spiny neurons
  • the functional neurons are mammalian neurons, eg, human, non-human primate, rat, mouse neurons.
  • Dopaminergic neurons are neurons that contain and release dopamine (DA) as the neurotransmitter.
  • Dopamine is a catecholamine neurotransmitter that plays an important biological role in the central nervous system.
  • Dopaminergic neurons in the brain are mainly concentrated in the substantia nigra pars compacta (SNc) of the midbrain and the ventral tegmentum. area (ventral tegmental area, VTA), hypothalamus and periventricular area.
  • SNc substantia nigra pars compacta
  • VTA ventral tegmental area
  • Many experiments have confirmed that dopaminergic neurons are closely related to various diseases in the human body, the most typical of which is Parkinson's disease.
  • Gene editing tools refer to the process of modifying target genes through gene editing technology, including the insertion, deletion or replacement of genes, thereby changing their genetic information and phenotypic characteristics.
  • available gene editing tools include, but are not limited to: CRIPSR gene editing tools (CRISPR/Cas system), zinc finger nuclease gene editing tools (ZFN system), TALENs (transcription activator-like (TAL) effector nucleases)) gene editing tools (TALEN system).
  • CRIPSR gene editing tools CRISPR/Cas system
  • ZFN system zinc finger nuclease gene editing tools
  • TALENs transcription activator-like (TAL) effector nucleases) gene editing tools
  • the gene editing tools include DNA gene editing tools or RNA gene editing tools.
  • the gene editing tool of the present disclosure is a CRIPSR gene editing tool.
  • the CRIPSR gene editing tool includes a nucleic acid encoding a Cas protein (also referred to as Cas enzyme herein) or a functional domain of the Cas protein, and a gRNA targeting a gene of interest. gRNA can guide the Cas protein to target the target gene and perform gene editing.
  • the Cas protein is Cas9, Cas12 (for example: Cas12a, Cas12b, Cas12c, Cas12d, etc.), Cas13, Cas 14, CasX or CasY family proteins or mutants thereof.
  • the Cas protein is Cas13a, Cas13d, Cas13X, Cas13a, Cas13b (for example: Cas13b-t1, Cas13b-t2, Cas13b-t3, etc.), Cas13c, or Cas13Y.
  • the Cas13d is CasRx.
  • the gene editing tool can reduce or silence the expression of the negative regulatory genes involved in this application.
  • gene editing tools can be delivered via vectors, for example, via adeno-associated viruses to deliver gRNAs and gene editing proteins (eg, CasRx) provided herein.
  • gRNAs and gene editing proteins eg, CasRx
  • the nucleotide of the Cas protein can be obtained through genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and its amino acid sequence can be derived from the nucleotide sequence.
  • the sources of the wild-type Cas protein include but are not limited to: Ruminococcus lavefaciens, Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae (Lachnospiraceaeacterium).
  • the Cas protein is capable of editing DNA.
  • the Cas protein is capable of editing RNA.
  • the Cas protein can be Cas13d, Cas13e, Cas13a, Cas13b, Cas13c, Cas13f and other RNA-targeting gene editing proteins.
  • protein of the present disclosure proteins
  • proteins proteins
  • polypeptide proteins
  • proteins are used interchangeably and may refer to RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, Protein or polypeptide of the amino acid sequence of PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, BTRC, CYP1B1, DPYSL2, BAF45b, SCF, HuB, HuC, or HuD. They include the protein with or without the starting methionine. Furthermore, the term also includes the full length of said protein and fragments thereof.
  • the proteins referred to in this disclosure include their complete amino acid sequences, their secreted proteins, their mutants, and their functionally active fragments.
  • RCOR1, RCOR2 and RCOR3 are REST corepressor 1, REST corepressor 2 and REST corepressor 3.
  • the three have similar functions; the full name of HDAC1 is Histone deacetylase 1 (Histone deacetylase 1), HDAC2 stands for Histone deacetylase 2 (Histone deacetylase 2), both of which have similar functions; KDM1A is called Lysine-specific demethylase 1A (Lysine-specific demethylase 1A), also known as Lysine-specific histone demethylase (Lysine -specific histone demethylase 1A, LSD1).
  • Sin3a member A of the SIN3 transcriptional regulatory protein family
  • Sin3b member B of the SIN3 transcriptional regulatory protein family
  • the terms “gene”, “polynucleotide” and “nucleic acid” are used interchangeably and may all refer to genes having RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a , Nucleic acid sequence of the nucleotide sequence of USP14, HuR, BrG1, EZH2, CDYL, HMG20B, CYP1B1, BTRC, DPYSL2, BAF45b, SCF, HuB, HuC, or HuD.
  • the full genome length of the human RCOR1 gene is 173,980 bp (NCBI GenBank accession number is 23186).
  • the full genome length of mouse RCOR1 gene is 76536bp (NCBI GenBank accession number is 217864).
  • the full genome length of the human RCOR2 gene is 5935bp (NCBI GenBank accession number is 283248).
  • the full genome length of mouse RCOR2 gene is 7832bp (NCBI GenBank accession number is 104383).
  • the full genome length of the human RCOR3 gene is 57020bp (NCBI GenBank accession number is 55758).
  • the full genome length of mouse RCOR3 gene is 39526bp (NCBI GenBank accession number is 214742).
  • the full genome length of the human Sin3a gene is 86441bp (NCBI GenBank accession number is 25942).
  • the full genome length of the mouse Sin3a gene is 56382 bp (NCBI GenBank accession number is 20466).
  • the full genome length of the human Sin3b gene is 50958 bp (NCBI GenBank accession number is 23309).
  • the full genome length of mouse Sin3b gene is 34934bp (NCBI GenBank accession number is 20467).
  • the full genome length of the human HDAC1 gene is 41546bp (NCBI GenBank accession number is 3065).
  • the full genome length of mouse HDAC1 gene is 26543bp (NCBI GenBank accession number is 433759).
  • the full genome length of the human HDAC2 gene is 38121bp (NCBI GenBank accession number is 3066).
  • the full genome length of the mouse HDAC2 gene is 27593 bp (NCBI GenBank accession number is 15182).
  • the full genome length of the human KDM1A gene is 64249bp (NCBI GenBank accession number is 23028).
  • the full genome length of the mouse KDM1A gene is 52284 bp (NCBI GenBank accession number is 99982).
  • the full genome length of the human PHF21A gene is 192136bp (NCBI GenBank accession number is 51317).
  • the full genome length of mouse PHF21A gene is 180916bp (NCBI GenBank accession number is 192285).
  • the full genome length of human BAF53a gene is 25482bp (NCBI GenBank accession number is 86).
  • the full genome length of the mouse BAF53a gene is 18428 bp (NCBI GenBank accession number is 56456).
  • the full genome length of the human G9a gene is 17940 bp (NCBI GenBank accession number is 10919).
  • the full genome length of the mouse G9a gene is 15624 bp (NCBI GenBank accession number is 110147).
  • the full genome length of the human USP14 gene is 56073 bp (NCBI GenBank accession number is 9097).
  • the full genome length of mouse USP14 gene is 36535bp (NCBI GenBank accession number is 59025).
  • the full genome length of human HuR gene is 47069bp (NCBI GenBank accession number is 1994).
  • the full genome length of mouse HuR gene is 40324bp (NCBI GenBank accession number is 15568).
  • the full genome length of the human BrG1 gene is 101,279 bp (NCBI GenBank accession number is 6597).
  • the full genome length of the mouse BrG1 gene is 88150 bp (NCBI GenBank accession number is 20586).
  • the full genome length of the human EZH2 gene is 76971bp (NCBI GenBank accession number is 2146).
  • the full genome length of the mouse EZH2 gene is 65102 bp (NCBI GenBank accession number is 14056).
  • the full genome length of the mouse CDYL gene is 214247 bp (NCBI GenBank accession number is 12593).
  • the full genome length of the human HMG20B gene is 249407bp (NCBI GenBank accession number is 9425) and 6168bp (NCBI GenBank accession number is 10362).
  • the full genome length of the mouse HMG20B gene is 4898 bp (NCBI GenBank accession number is 15353).
  • the full genome length of the human DPYSL2 gene is 144145bp (NCBI GenBank accession number is 1808).
  • the full genome length of the mouse DPYSL2 gene is 108178 bp (NCBI GenBank accession number is 12934).
  • the full genome length of the human BAF45b gene is 18690 bp (NCBI GenBank accession number is 8193).
  • the full genome length of the mouse BAF45b gene is 13652 bp (NCBI GenBank accession number is 29861).
  • the full genome length of the human SCF gene is 87679 bp (NCBI GenBank accession number is 4254).
  • the full genome length of the mouse SCF gene is 84802bp (NCBI GenBank accession number is 17311).
  • the full genome length of the human HuB gene is 160503 bp (NCBI GenBank accession number is 1993).
  • the full genome length of the mouse HuB gene is 188638 bp (NCBI GenBank accession number is 15569).
  • the full genome length of the human HuC gene is 29721bp (NCBI GenBank accession number is 1995).
  • the full genome length of the mouse HuC gene is 37142 bp (NCBI GenBank accession number is 15571).
  • the full genome length of the human HuD gene is 155718 bp (NCBI GenBank accession number is 1996).
  • the full genome length of the mouse HuD gene is 148212 bp (NCBI GenBank accession number is 15572).
  • the full genome length of the human CYP1B1 gene is 8643bp (NCBI GenBank accession number is 1545).
  • the full genome length of mouse CYP1B1 gene is 8122bp (NCBI GenBank accession number is 13078).
  • the full genome length of the human BTRC gene is 203506bp (NCBI GenBank accession number is 8945).
  • the full genome length of the mouse BTRC gene is 169664 bp (NCBI GenBank accession number is 12234).
  • the similarity between human and mouse RCOR1 at the DNA level is 88.51%; the protein sequence similarity is 92.39%.
  • the similarity between human and mouse RCOR2 at the DNA level is 89.26%; the protein sequence similarity is 97.51%.
  • the similarity between human and mouse RCOR3 at the DNA level is 92.38%; the protein sequence similarity is 96.39%.
  • the similarity between human and mouse Sin3a at the DNA level is 90.91%; the protein sequence similarity is 98.04%.
  • the similarity between human and mouse Sin3b at the DNA level is 86.61%; the protein sequence similarity is 87.91%.
  • the similarity between human and mouse HDAC1 at the DNA level is 90.85%; the protein sequence similarity is 99.38%.
  • the similarity between human and mouse HDAC2 at the DNA level is 91.62%; the protein sequence similarity is 99.59%.
  • the similarity between human and mouse KDM1A at the DNA level is 89.75%; the protein sequence similarity is 97.72%.
  • the similarity between human and mouse PHF21A at the DNA level is 92.88%; the protein sequence similarity is 94.20%.
  • the similarity between human and mouse BAF53a at the DNA level is 88.60%; the protein sequence similarity is 98.84%.
  • the similarity between human and mouse G9a at the DNA level is 87.74%; the protein sequence similarity is 94.75%.
  • the similarity between human and mouse USP14 at the DNA level is 91.31%; the protein sequence similarity is 96.76%.
  • the similarity between human and mouse HuR at the DNA level is 91.03%; the protein sequence similarity is 98.47%.
  • the similarity between human and mouse BrG1 at the DNA level is 89.00%; the protein sequence similarity is 95.47%.
  • the similarity between human and mouse EZH2 at the DNA level is 91.98%; the protein sequence similarity is 97.60%.
  • the similarity between human and mouse CDYL at the DNA level is 86.77%; the protein sequence similarity is 93.12%.
  • the similarity between human and mouse HMG20B at the DNA level is 85.53%; the protein sequence similarity is 93.69%.
  • the similarity between human and mouse DPYSL2 at the DNA level is 91.05%; the protein sequence similarity is 97.64%.
  • the similarity between human and mouse BAF45b at the DNA level is 93.39%; the protein sequence similarity is 97.69%.
  • the similarity between human and mouse SCF at the DNA level is 89.44%; the protein sequence similarity is 82.78%.
  • Human and mouse HuB in DNA water The similarity is 95.46%; the protein sequence similarity is 99.72%.
  • the similarity between human and mouse HuC at the DNA level is 91.94%; the protein sequence similarity is 99.46%.
  • the similarity between human and mouse HuD at the DNA level is 94.64%; the protein sequence similarity is 99.21%.
  • the similarity between human and mouse CYP1B1 at the DNA level is 82.96%; the protein sequence similarity is 81.03%.
  • nucleic acid sequence encoding it can be constructed based on it, and a specific probe can be designed based on the nucleotide sequence.
  • the full-length nucleotide sequence or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • primers can be designed based on the nucleotide sequences disclosed in the present disclosure, especially the open reading frame sequence, and commercially available cDNA libraries or cDNA prepared by conventional methods known to those skilled in the art can be used
  • the library is used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • recombination can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.
  • artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is short. Often, fragments with long sequences are obtained by first synthesizing multiple small fragments and then ligating them.
  • DNA sequence encoding the protein of the present disclosure can be obtained entirely through chemical synthesis.
  • the DNA sequence can then be introduced into a variety of existing DNA molecules (eg, vectors) and cells known in the art.
  • polynucleotide sequences of the present disclosure may be used to express or produce recombinant polypeptides by conventional recombinant DNA techniques. Generally speaking there are the following steps:
  • polynucleotide sequences can be inserted into recombinant expression vectors.
  • any plasmid and vector can be used as long as it can replicate and be stable in the host body.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translation control elements.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green color for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green color for eukaryotic cell culture.
  • GFP Fluorescent protein
  • tetracycline or ampicillin resistance in E. coli tetracycline or ampicillin resistance in E. coli.
  • Vectors containing appropriate DNA sequences as described above and appropriate promoter or control sequences can be used to transform appropriate host cells to enable expression of proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, using steps well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present disclosure.
  • the medium used in culture can be selected from various conventional media. Cultivate under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced using an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed within the cell, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Adeno-associated virus is smaller than other viral vectors, non-pathogenic, and can transfect both dividing and undividing cells, gene therapy methods for genetic diseases based on AAV vectors have received considerable attention. Widespread concern.
  • Adeno-associated virus also known as adeno-associated virus, belongs to the genus Parvoviridae and is a type of single-stranded DNA defective virus with the simplest structure currently discovered. It requires a helper virus (usually an adenovirus) to participate in replication. It encodes the cap and rep genes in two terminal inverted repeats (ITRs). ITRs play a decisive role in virus replication and packaging. The cap gene encodes the viral capsid protein, and the rep gene is involved in virus replication and integration. AAV can infect a variety of cells.
  • Recombinant adeno-associated virus vector is derived from non-pathogenic wild-type adeno-associated virus. Due to its good safety, wide range of host cells (dividing and non-dividing cells), and low immunogenicity, it can express foreign genes in vivo for a long time. With long and other characteristics, it is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research around the world. After more than 10 years of research, the biological properties of recombinant adeno-associated viruses have been deeply understood, especially their application effects in various cells, tissues and in vivo experiments. A lot of data has been accumulated.
  • rAAV is used in gene therapy research for a variety of diseases (including in vivo and in vitro experiments); at the same time, as a unique gene transfer vector, it is also widely used in gene function research, construction of disease models, and gene preparation. aspects such as knockout mice.
  • the vector is a recombinant AAV vector.
  • AAVs are relatively small DNA viruses that integrate into the genome of the cells they infect in a stable and site-specific manner. They are able to infect a large range of cells without any effect on cell growth, morphology or differentiation, and they do not appear to be involved in human pathology.
  • the AAV genome has been cloned, sequenced and characterized.
  • AAV contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the virus's origin of replication. The remainder of the genome is divided into two important regions with encapsidation functions: the left portion of the genome containing the rep genes involved in viral replication and viral gene expression; and the right portion of the genome containing the cap gene encoding the viral capsid protein.
  • ITR inverted terminal repeat
  • AAV vectors can be prepared using standard methods in the art. Adeno-associated virus of any serotype is suitable. Methods for purifying vectors can be found, for example, in U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described, for example, in PCT Application No. PCT/US2005/027091, the disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors for gene transport in vitro and in vivo has been described (see, for example, International Patent Application Publication Nos. WO91/18088 and WO93/09239; U.S. Patent Nos.
  • Replication-deficient recombinant AAVs can be prepared by co-transfecting a plasmid containing a nucleic acid sequence of interest flanked by two AAV inverted terminal repeats (ITRs) into a cell line infected with a human helper virus (eg, adenovirus). region, and a plasmid carrying AAV encapsidation genes (rep and cap genes). The resulting AAV recombinants are then purified by standard techniques.
  • ITRs AAV inverted terminal repeats
  • the recombinant vector is encapsidated into viral particles (eg, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16 AAV virions).
  • viral particles eg, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16 AAV virions.
  • the present disclosure includes recombinant virions (recombinant because they contain recombinant polynucleotides) containing any of the vectors described herein. Methods of producing such particles are known in the art and are described in U.S. Patent No. 6,596,535.
  • the AAV vector may be modified to include a modified VP protein (e.g., modified to include a VP1 protein, AAV vector of VP2 protein or VP3 protein).
  • the AAV vector is a recombinant AAV (rAAV) vector.
  • rAAV may consist of a capsid sequence and structure that is substantially similar to that found in wild-type AAV (wtAAV).
  • wtAAV wild-type AAV
  • rAAV encapsulates a genome that is essentially devoid of AAV protein coding sequences and has a therapeutic gene expression cassette designed in its place, such as the subject polynucleotide.
  • virally derived sequences may be ITRs that may be required to direct genome replication and packaging during vector production.
  • Suitable AAV vectors may be selected from any AAV serotype or combination of serotypes.
  • the AAV vector can be any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15, AAV 16, AAV .rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5 , AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HS
  • the vector is selected based on its natural orientation. In some cases, the vector serotype is selected based on its ability to cross the blood-brain barrier. AAV9 and AAV10 have been shown to cross the blood-brain barrier to transduce neurons and glial cells. In one aspect, the AAV vector is AAV2, AAV5, AAV6, AAV8 or AAV9. In some cases, the AAV vector is a chimera of at least two serotypes. In one aspect, the AAV vectors are serotypes AAV2 and AAV5. In some cases, chimeric AAV vectors include rep and ITR sequences from AAV2 and cap sequences from AAV5.
  • chimeric AAV vectors include rep and ITR sequences from AAV2 and cap sequences from any other AAV serotype.
  • AAV vectors may be self-complementary.
  • AAV vectors may include inverted terminal repeats.
  • AAV vectors may include inverted terminal repeat (scITR) sequences with mutated terminal melting sites.
  • rep, cap and ITR sequences from all the different AAV serotypes provided herein can be mixed and matched.
  • the AAV vector is from an adeno-associated virus with a serotype selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14 , AAV 15, AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV .PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV .HSC9, AAV.HSC10, AAV.HSC11
  • the vector can be a recombinant AAV (rAAV) vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementing AAV (scAAV) vector, a single-stranded AAV, or any combination thereof.
  • the AAV vector includes a genome comprising replicated genes and inverted terminal repeats from a first AAV serotype and capsid proteins from a second AAV serotype.
  • the AAV vector may be chimeric and may be: an AAV2/5 vector, an AAV2/6 vector, an AAV2/7 vector, an AAV2/8 vector, or AAV2/9 vector.
  • the inverted terminal repeats of the AAV vector include 5' inverted terminal repeats, 3' inverted terminal repeats, and mutated inverted terminal repeats. In some cases, the mutated inverted terminal repeat lacks a terminal melting site.
  • suitable AAV vectors can be further modified to encompass modifications such as in the capsid or rep protein. Modifications may also include deletions, insertions, mutations, and combinations thereof.
  • the carrier is modified to reduce immunogenicity, allowing for repeated administration. In some cases, when repeated administration is performed to reduce and/or eliminate immunogenicity, the serotype of the vector used changes.
  • the inhibitor is one or several types of negative regulators, which inhibit the expression of negative regulatory genes, or inhibit the mRNA levels of negative regulatory genes, or inhibit the levels of encoded proteins of negative regulatory genes.
  • various conventional screening methods can be used to screen out gene expression levels, or mRNA levels, or protein expression levels, or proteins that can enhance DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC. Positive regulator of activity.
  • the enhancer agent is one or several types of positive regulators that enhance the expression of positively regulated genes, or enhance the mRNA levels of positively regulated genes, or enhance the levels of encoded proteins of positively regulated genes.
  • Negative or positive modulators useful in the present disclosure may be substances that reduce, eliminate, or eliminate the expression and/or activity of the gene, its RNA (eg, mRNA), or its encoded protein at the DNA, RNA, or protein level.
  • the negative regulator includes inhibitory antibodies of negatively regulated genes; or small molecule inhibitors of negatively regulated genes; or inhibitory mRNA, microRNA, siRNA, shRNA, antisense oligos of negatively regulated genes. Nucleotides, binding proteins or protein domains, polypeptides, nucleic acid aptamers, or PROTACs; or inhibitory binding proteins or ligands that negatively regulate genes.
  • negative modulators of RCOR1 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO: 68.
  • Negative modulators of RCOR2 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:69.
  • Negative modulators of RCOR3 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:70.
  • Negative modulators of Sin3a of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:71.
  • Negative modulators of Sin3b of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:72.
  • Negative modulators of HDAC1 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:73.
  • Negative modulators of KDM1A of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:74.
  • Negative modulators of HDAC2 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:75.
  • Negative modulators of PHF21A of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:76.
  • the present disclosure negatively regulates BAF53a
  • Modulating agents include inhibitors targeting a target sequence comprising SEQ ID NO:77.
  • Negative modulators of G9a of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:78.
  • Negative modulators of USP14 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:79.
  • Negative modulators of HuR of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:80.
  • Negative modulators of BrG1 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:81.
  • Negative modulators of EZH2 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:82.
  • Negative modulators of CDYL of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:83.
  • Negative modulators of HMG20B of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:84.
  • the targets of the negative regulator or positive regulator of the present disclosure include astrocytes or MG cells.
  • the positive regulator can be a substance that increases the expression and/or activity of a positively regulated gene, or its RNA (such as mRNA), or its encoded protein at the DNA, RNA, or protein levels.
  • positive regulators include: expression vectors containing promoters, endogenous expression activators, protein analogs or enhancers, epigenetic regulatory tools that can improve the expression of positively regulated genes, positively regulated genes Expression activators, degradation inhibitors of proteins encoded by positive regulatory genes, stabilizers of positive regulatory gene mRNA, or exogenous positive regulatory genes or functional fragments of positive regulatory genes.
  • the methods and steps of negative regulator regulation include using antibodies to neutralize its proteins, using shRNA or siRNA carried by viruses (such as adeno-associated viruses), or using gene editing tools to silence genes.
  • the inhibition rate of negative regulators on negatively regulated genes is generally at least 50% or more, preferably 60%, 70%, 80%, 90%, or 95% or more.
  • the inhibition rate can be controlled and detected based on conventional techniques, such as flow cytometry, fluorescence quantitative PCR or Western blot.
  • Negative regulators including antibodies, antisense nucleic acids, gene editing tools and other inhibitors
  • positive regulators including: expression vectors containing promoters, endogenous expression activators, protein analogs or Enhancer
  • these materials when administered therapeutically, can inhibit or enhance the expression and/or activity of the gene or protein, thereby inducing the differentiation of glial cells into neuronal cells, thereby treating neuronal dysfunction. or death-related illness.
  • these materials may be formulated in a nontoxic, inert, and pharmaceutically acceptable aqueous medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary with the materials being formulated. It varies depending on the nature of the condition and the condition to be treated.
  • the prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): topical, intramuscular, intracranial, intraocular, intraperitoneal, intravenous, subcutaneous, intradermal, local administration, autologous Cells are extracted and cultured and then reinfused.
  • the present disclosure also provides a pharmaceutical composition, which contains a safe and effective amount of the negative modulator or positive modulator of the present disclosure and a pharmaceutically acceptable carrier or excipient.
  • Such carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present disclosure can be formulated into an injection form, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Make preparations.
  • Pharmaceutical compositions, such as tablets and capsules can be prepared by conventional methods.
  • Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, about 1 microgram to 10 mg/kg of body weight per day.
  • This disclosure finds for the first time that RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1,
  • EZH2, CDYL, or HMG20B genes or their encoded proteins and/or increasing the expression of DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC genes or their encoded proteins in glial cells or activity, which can induce the differentiation of glial cells into dopamine neuron cells, thereby preventing and/or treating diseases related to neuronal function loss or death.
  • DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC in glial cells, and directly transform Muller cells into RGC cells or photoreceptor cells.
  • Regenerated RGCs can be integrated into the visual pathway and improve visual function in RGC injury mouse models.
  • nSR100 In order to overexpress nSR100 in glial cells, the glial cell-specific promoter GFAP was selected to initiate the expression of nSR100.
  • Transient transfection of astrocytes and qPCR Isolate primary mouse astrocytes and seed astrocytes in 6-well plates. Transient transfection was performed with 3 ⁇ g of vector expressing gRNA-CasRx-GFP using Lipofectamine 3000 (Thermo Fisher Scientific) according to standard procedures. Control plasmid expresses non-targeting gRNA. 1-2 days after transient transfection, GFP-positive cells were collected by flow cytometric cell sorting (FACS) and lysed for qPCR analysis.
  • FACS flow cytometric cell sorting
  • AAV8 was used to carry the gene of interest.
  • the titer of AAV-CasRx, AAV-CasRx-gRNA, or AAV-GFAP-positive regulatory gene is greater than 5 ⁇ 10 12 vg/ml (1-3 ⁇ l per injection).
  • AAV was injected into the striatum (AP+0.8mm, ML ⁇ 1.6mm and DV-2.8mm).
  • Immunofluorescence staining Mice were perfused 6-12 weeks after injection, and brains were removed and fixed with 4% paraformaldehyde (PFA) overnight and dehydrated in 30% sucrose for at least 12 hours. After embedding, the sections were frozen and sectioned to a thickness of 30 ⁇ m. Before immunofluorescence staining, brain sections were rinsed thoroughly with 0.1 M phosphate buffered saline (PBS). Primary antibodies: rabbit polyclonal NeuN antibody (Brain, 1:500, #ABN78, Millipore), mouse TH antibody (1:300, MAB318, Millipore).
  • RNA-seq 293T or N2a cells were cultured in 15-cm dishes and transiently transfected with 70 ⁇ g of plasmid. The first 20% of GFP and mCherry double-positive N2a cells were collected by FACS, RNA was extracted, converted to cDNA, and then used for whole-transcriptome RNA-seq.
  • Intravitreal and subretinal injections were introduced via intravitreal and subretinal injections, respectively, as previously described.
  • Subretinal injection Inject into the eye using a Hamilton syringe (32G needle) under an Olympus microscope AAV (>1 ⁇ 10 13 vg/ml). To determine reprogramming in intact retinas, a total of 1 ⁇ l of GFAP-GFP-Cre (0.2 ⁇ l) and AAV-GFAP-nSR100, or GFAP-CasRx-gRNA (0.8 ⁇ l), or GFAP-GFP-Cre (0.2 ⁇ l) and GFAP-CasRx (0.8 ⁇ l) were delivered to the retina.
  • Immunofluorescence staining 1.5-3 months after AAV injection, the eyes, optic nerve and brain were removed and fixed with 4% paraformaldehyde (PFA) for 2 hours (eyes and optic nerve) or 24 hours (brain), and then incubated in 30% sucrose. Dehydrate in solution for 2 hours (eyes and optic nerves) or 24 hours (brain). After embedding and freezing, eyes and brains were sectioned at a thickness of 30 ⁇ m.
  • PFA paraformaldehyde
  • gRNAs targeting RCOR1, Sin3a, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, and HMG20B were designed, and CasRx and gRNA were co-administered. Transfect N2A cells.
  • nucleotide sequence and target sequence of gRNA are shown in Table 1 above.
  • the transfection method is: transiently transfect 4 ⁇ g CAG-CasRx-P2A-GFP plasmid and 2 ⁇ g U6-gRNA-CMV-mCherry plasmid to determine the inhibitory effect of each gRNA in in vitro cell lines. Use CAG-CasRx-P2A-GFP plasmid alone. Transfection served as control. Lipofectamine 3000 (Thermo Fisher Scientific) was used according to standard procedures. Two days after transfection, 30,000 GFP and mCherry double-positive cells were collected from each sample by fluorescence-activated cell sorting (FACS), and lysed for qPCR analysis. See Table 2 for qPCR primers.
  • FACS fluorescence-activated cell sorting
  • Example 2 Study on the transdifferentiation of astrocytes into neurons by negative regulatory genes
  • AAV-GFAP was injected into the striatum of C57BL/6 mice older than eight weeks.
  • the plasmid design is shown in Figure 2A and B.
  • Figure 4 shows the statistics of astrocytes converted into neurons after knocking down different negative regulatory genes. Among them, RCOR1, G9a, BAF53A, etc. are more efficient in inducing glial cells to transdifferentiate into neurons.
  • Example 3 Study on the transdifferentiation of astrocytes into neurons by positive regulatory genes
  • AAV in the control or test group was injected into the striatum or substantia nigra of mice, in which vector 1 is AAV-GFAP-mCherry, and vector 2 (AAV-GFAP-Gene) is driven by the astrocyte-specific promoter GFAP.
  • Target gene expression Similar to the knockdown target gene expression group, samples were taken 1-3 months after AAV injection to analyze the efficiency of different targets in inducing glial cells to transdifferentiate into neurons. The results are shown in Figures 5 and 6.
  • Example 7 In order to further study whether the overexpression of positive regulatory genes and the inhibition of negative regulatory genes can promote the transdifferentiation of astrocytes into dopamine neurons, referring to the operations of Example 1 to Example 3, we injected AAV in the control group or test group was stained with the dopamine neuron-specific protein marker TH. The results are shown in Figure 7. No evidence was found in the control group. TH-positive cells ( Figure 7A), TH-positive dopamine neurons co-labeled with mCherry signals were found in the HuB and BTRC overexpression group, indicating that overexpression of HuB and BTRC can induce astrocytes to transform into dopamine neurons. differentiation.
  • Müller glia cells perform functions similar to glial cells, and the retina contains a variety of neurons with specialized functions, such as photoreceptor cells and bipolar cells.
  • GFAP-EGFP-2A-Cre system combined with Ai9 mice to achieve specific labeling of Müller glia.
  • the schematic diagram is shown in Figure 8. Referring to the operations of Examples 1 to 3, AAV-GFAP-CasRx-gRNA (or A mixed virus of control vector AAV-GFAP-CasRx) and AAV-GFAP-EGFP-2A-Cre.
  • AAV-GFAP-EGFP-2A-Cre is used to specifically label Müller glial cells or transformed from glial cells.
  • Neurons (retinal ganglion cells or photoreceptor cells), 1-3 months after injection, the retina is taken for slice analysis to study whether the corresponding negative regulatory genes are inhibited or the corresponding positive regulatory genes are upregulated.
  • Müller glia transdifferentiate into photoreceptor cells.
  • GFAP-EGFP-2A-Cre+GFAP-CasRx was used as the control group, and GFAP-EGFP-2A-Cre+GFAP-CasRx-gRNA was used as the test group.
  • the gRNA targeted Sin3a, HDAC2, BAF53a, G9a, HuR, BrG1, EZH2, CDYL, and KDM1A.
  • GFAP-EGFP-2A-Cre was used as the control group, and GFAP-EGFP-2A-Cre+GFAP-positive regulatory genes were used as the test group, among which the positive regulatory genes were DPYSL2, BAF45b, SCF, CYP1B1, and BTRC genes.
  • FIG. 9A The effects of negative regulatory genes on transdifferentiation are shown in Figure 9.
  • Figure 9A in the GFAP-EGFP-2A-Cre group, Müller glial cells showed a typical morphology, with the cell body located in the inner granular layer (INL) and processes distributed to the outer granular layer (ONL) and retinal ganglion cell layer. (GCL), and no red signal-labeled photoreceptor cells were observed in the ONL, indicating that there would be no transdifferentiation of Müller glia into photoreceptor cells in the control group.
  • CasRx knocks down the expression of negative regulatory genes or overexpresses positive regulatory genes to reprogram Müller glia into photoreceptor cells.
  • the quantitative statistics are shown in Figure 11, showing inhibition of Sin3a, KDM1A, BAF53a, G9a, HuR , BrG1, EZH2, and CDYL all show excellent photoreceptor cell regeneration ability.
  • Inhibiting HDAC2 also has photoreceptor cell regeneration ability, and overexpressing DPYSL2 and BAF45b also has photoreceptor cell regeneration ability.
  • Photoreceptor cells are divided into cones and rods.
  • Sin3a and KDM1A groups to use rod-specific Staining with sex protein markers, as shown in Figure 12, found that most of the photoreceptor cells transdifferentiated from Müller glia cells in the ONL layer were rod cells.
  • Müller glia can be transdifferentiated into retinal ganglion cells (RGC), subretinal injection of different AAV combinations into NMDA-modeled 6-8 week Ai9 (Rosa26-CAG-LSL-tdTomato-WPRE) mice .
  • RRC retinal ganglion cells
  • AAV-GFAP-CasRx-gRNA or control vector AAV-GFAP-CasRx
  • AAV-GFAP-EGFP-2A-Cre is used to specifically label Müller glia or neurons transformed from glial cells (optic ganglion cells).
  • AAV-GFAP-EGFP-2A-Cre was injected as the control group, and AAV-GFAP-EGFP-2A-Cre+AAV-GFAP-positive regulatory genes were injected (the positive regulatory genes were DPYSL2, BAF45b, SCF, HuB , HuC, HuD, CYP1B1, BTRC).
  • the number of cells and the statistical chart of the number of tdTomato-positive axons in the optic nerve are shown in Figure 16. More optic ganglion cells were found in the SCF, HuD, and CYP1B1 groups.
  • the Pde6b gene is a disease-causing gene related to retinitis pigmentosa (RP). Defects in the Pde6b gene can lead to the death of photoreceptor cells.
  • RP retinitis pigmentosa
  • Pde6b gene-deficient mice have been proven to be disease animal models with genetic characteristics of RP.
  • a Pde6b gene knockout mouse model constructed based on CRISPR-Cas9 editing was used to prove through immunohistochemical methods In Pde6b knockout mice, a large number of rod cells died at 4 weeks after birth, and all rod cells died at 6 weeks ( Figure 17A-C).
  • AAV AAV-U6-gRNA(KDM1A)-GFAP-CasRx and AAV-U6-gRNA(EZH2)-GFAP-CasRx
  • KDM1A and EZH2 transdifferentiation targets
  • EZH2 transdifferentiation targets
  • AAV-CBH-Pde6b AAV overexpressing Pde6b
  • the control group was simultaneously injected with control AAV (AAV-GFAP-CasRx) and AAV overexpressing Pde6b (AAV-CBH-Pde6b).
  • the injection volume of transdifferentiation target AAV The injection volume of Pde6b overexpressing AAV was 1x10 10 vg, and the injection volume was 5x10 9 vg, and the total volume of injected AAV was 1 ⁇ l.
  • retinal tissue was taken for analysis. It was found that there were no photoreceptor cells in the outer granular layer of the retina of mice injected with AVV (AAV-GFAP-CasRx+AAV-CBH-Pde6b) in the control group, using rod-specific markers. Rhodopsin was stained, and no Rhodopsin-positive cells were found (Figure 17D).
  • Example 3 and Example 4 the applicant described in detail the results of injecting the negative regulator or the positive regulator of the present application into the striatum.
  • Those skilled in the art can refer to the results of the present application.
  • Technical solutions and common knowledge in the field can choose to perform positioning injections in other parts of the brain, such as targeting the ventral tegmental area of the midbrain, spinal cord, hypothalamus, dorsal midbrain, cerebral cortex, hippocampus, cerebellum, or Injections into the substantia nigra, especially injections into the substantia nigra, also have the same or similar technical effects.
  • Example 5 the applicant described in detail the results of subretinal injection of the negative regulator or positive regulator of the present application.
  • Those skilled in the art can rely on the technical solutions of the present application and the According to conventional knowledge, you can choose to perform positioning injections in other parts of the eye, such as the vitreous cavity, which will also have the same or similar technical effects.

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Abstract

L'invention concerne un procédé de trans-différenciation de cellules non neuronales d'un mammifère en neurones. Le procédé consiste à obtenir un régulateur négatif pouvant réduire l'expression des gènes régulateurs négatifs, le gène régulateur négatif comprenant RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, ou HMG20B, et/ou à obtenir un régulateur positif pouvant améliorer l'expression des gènes régulateurs positifs, les gènes régulateurs positifs comprenant DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, ou BTRC. Une quantité efficace du régulateur négatif ou du régulateur positif en contact avec les cellules non neuronales peut induire une trans-différenciation des cellules animales non neuronales en neurones.
PCT/CN2023/115930 2022-08-30 2023-08-30 Procédé de trans-différenciation de cellules non neuronales en neurones et son utilisation WO2024046393A1 (fr)

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